Molecular Membrane Biology, April /June 2003, 20, 141 /154
Membrane dynamics and the biogenesis of lysosomes (Review)
J. Paul Luzio$*, Viviane Poupon$, Margaret
R. Lindsay$, Barbara M. Mullock$, Robert C. Piper% and
Paul R. Pryor$
$ Cambridge Institute for Medical Research and Department
of Clinical Biochemistry, University of Cambridge,
Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2XY,
UK
% Department of Physiology and Biophysics, University of
Iowa, Iowa City, IA 52242, USA
Summary
Lysosomes are dynamic organelles receiving membrane traffic
input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid
hydrolases and are capable of fusing with late endosomes to
form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid
organelles involves content condensation and probably
removal of some membrane proteins by vesicular traffic.
Lysosomes can also fuse with the plasma membrane in
response to cell surface damage and a rise in cytosolic Ca2
concentration. This process is important in plasma membrane
repair. The molecular basis of membrane traffic pathways
involving lysosomes is increasingly understood, in large part
because of the identification of many proteins required for
protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been
identified and studied in the processes of vesicular delivery
of newly synthesized lysosomal proteins from the trans-Golgi
network, fusion of lysosomes with late endosomes and sorting
of membrane proteins into lumenal vesicles. Several multiprotein oligomeric complexes required for these processes
have been identified. The present review focuses on current
understanding of the molecular mechanisms of fusion of
lysosomes with both endosomes and the plasma membrane
and on the sorting events required for delivery of newly
synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.
Keywords: Endocytosis; multivesicular body; endosome /lysosome fusion; membrane traffic; organelle biogenesis..
Introduction
Lysosomes have long been regarded as the terminal
degradative compartment of the endocytic pathway in animal
cells. They are defined as membrane bound organelles of /
0.5 mm diameter containing a variety of acid hydrolases.
Their limiting membrane is enriched in integral membrane
glycoproteins, variously described as lysosome associated
membrane proteins (lamps), lysosome integral membrane
proteins (limps) or lysosome glycoproteins (lgps). Lysosomes can be distinguished from endosomes by the lack of
*To whom correspondence should be addressed.
E-mail: [email protected]
mannose 6-phosphate receptors (MPRs) and recycling
plasma membrane receptors (Kornfeld and Mellman 1989,
Luzio et al . 2000, Mullins and Bonifacino 2001). They have
an heterogenous morphology when observed by EM, often
appear electron dense by comparison with cytosol and
contain internal, i.e. lumenal, membrane vesicles and/or
membrane whirls (Holtzmann 1989). Lysosomes typically
constitute 0.5 /5% of the cell volume and are concentrated
near microtubule organizing centres (Matteoni and Kreis
1987). In both plant cells and yeast, the vacuole may be
regarded as the functional equivalent of the lysosome
(Klionsky et al . 1990, Klionsky 1997). In recent years,
genetic studies of the yeast vacuole and of the biogenesis
and function of lysosome-related organelles in animal cells
have led to the identification of many proteins involved in the
biogenesis and function of both these organelles and of
lysosomes. Lysosome-related organelles in animal cells that
have been particularly important in this respect include the
pigment granules in the Drosophila eye, melanosomes in
skin melanocytes and secretory lysosomes in cells of
haematopoietic origin. All of these organelles are like
lysosomes in that they contain acid hydrolases and lamps
but no MPRs and are accessible to endocytosed markers, at
least during their formation. They also contain specific
proteins not found in conventional lysosomes.
The discovery of lysosomes
Lysosomes were originally discovered by de Duve in 1949,
as a result of the latency of acid phosphatase activity in subcellular fractions and their role in the proteolysis of endocytosed macromolecules, in phagocytosis and in autophagy,
was rapidly established (reviewed in Bainton 1981, de Duve
1983). In addition, mainly from experiments in protozoa, it
was recognized that lysosomes could eliminate undigested
residues by fusion with the plasma membrane. Although by
the time of her review, Bainton (1981) had drawn the
conclusion that ‘the lysosomal system is not just a garbage
dump’, a rather static view of the lysosome as a terminal
degradation compartment or garbage disposal unit was
widely held until recent years. Two developments in particular have changed this perception. First, the realization that
lysosomes interact with late endosomes by undergoing many
‘kiss and run’ events and/or direct fusion and, secondly, that
both lysosome-related organelles called secretory lysosomes and conventional lysosomes can fuse with the plasma
membrane in response to a rise in cytosolic calcium
concentration.
Lysosomes as dynamic organelles
The overall route map of the endocytic pathway (Figure 1) is
well understood with delivery of endocytosed macromole-
Molecular Membrane Biology
ISSN 0968-7688 print/ISSN 1464-5203 online # 2003 Taylor & Francis Ltd
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DOI: 10.1080/0968768031000089546
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Figure 1. A model of endocytic biosynthetic and autophagic routes
of delivery to lysosomes. Arrows indicate traffic routes between
compartments. In this model direct fusion between late endosomes
and lysosomes is shown, but see Figure 2 for alternative methods of
delivering endosomal content to lysosomes.
cules first to early endosomes, then late endosomes and
finally lysosomes (reviewed in Mellman 1996, Mukherjee et
al . 1997, Marsh 2001). Delivery from late endosomes to
lysosomes has been the subject of considerable experimental investigation, testing different hypotheses (Figure 2).
Whilst some maturation events undoubtedly do occur in the
late endocytic pathway (see below), maturation of endo-
Figure 2. Diagrammatic representation of hypotheses to explain
delivery of endocytozed macromolecules from late endosomes to
lysosomes.
somes to lysosomes (Murphy 1991) is no longer regarded as
the main, or indeed likely, means of delivery by most
commentators. Similarly, whilst vesicular transport is clearly
of major importance in transferring content between donor
and acceptor organelles elsewhere in secretory and endocytic pathways (Mellman and Warren 2000), there is little
evidence for vesicular traffic between late endosomes and
lysosomes. To explain the size dependent, differential
transfer of newly endocytosed molecules into lysosomes
and observations from living cells in which labelled late
endocytic organelles continuously bump into each other,
Storrie and Desjardins (1996) proposed the ‘kiss and run’
hypothesis of lysosome biogenesis. The occurrence of
complete fusion, possibly developing from initial kisses,
was demonstrated following the development of cell-free
assays to study content mixing between endosomes and
lysosomes derived from rat hepatocytes (Mullock et al .
1998). The product of content mixing was found to reside
in a hybrid organelle, which was intermediate in density
between late endosomes and lysosomes and shown by EM
to contain markers derived from each starting organelle. The
discovery of this hybrid organelle is consistent with a view of
lysosome function in the endocytic pathway in which lysosomes act as storage organelles for acid hydrolases and
fuse with late endosomes to create a digestive organelle or
‘cell stomach’ (Griffiths 1996). Thus, not withstanding the
presence of some active hydrolases in late endosomes
(Casciola-Rosen and Hubbard 1991, Pillay et al . 2002), the
hybrid organelle may act as the major site for hydrolysis of
endocytosed macromolecules. Lysosomes are reformed
from the hybrid organelles by a maturation process which
includes condensation of content and the removal of some
membrane proteins and soluble content by vesicular carriers
(Pryor et al . 2000a). An important point to note is that,
according to the definitions of lysosomes and endosomes
based on the absence or presence of MPRs, a newly formed
hybrid organelle, where digestion of endocytosed macromolecules by lysosomal hydrolases will commence, is by
definition an endosome, whereas at some point in the
maturation process for the reformation of a lysosome it will
have lost its MPRs and be defined as a lysosome.
In some cell types, there is evidence that the lysosomal
compartment is modified to contain cell type-specific proteins
that are subject to regulated secretion. The resulting Ca2
regulated secretory granules include the lytic granules in
cytotoxic T cells and natural killer cells, dense granules in
platelets and histamine-containing granules in mast cells.
They are collectively referred to as secretory lysosomes
which serve as both degradative and secretory compartments and are most usually found in cells of the haematopoietic lineage. A more extensive list of these organelles and
their properties is found in the excellent review by Blott and
Griffiths (2002). Often included amongst secretory lysosomes are the storage compartments in antigen presenting
cells, i.e. macrophages, B cells and dendritic cells. These are
known as MIICs, which store major histocompatibility complex Class II molecules. They also contain endocytosed
antigen (Harding et al . 1991). Activation of dendritic cells
results in processing and conversion of the antigens to
peptide-MHC II complexes and causes tubules and vesicles
Lysosome biogenesis
to emerge from MIICs in order to deliver these complexes to
the cell surface (Barois et al . 2002, Chow et al . 2002).
Recently, a regulated secretory function for conventional
lysosomes has also been recognized. There is evidence that,
in many cell types, lysosomes and lysosome-related organelles can repair plasma membrane damage occurring in
response to experimental, physiological or pathological
trauma (Andrews 2000, Woolley and Martin 2000, McNeil
and Terasaki 2001, McNeil 2002). In sea urchin eggs, for
example, the surface tear resulting from removal of more
than 1000 mm2 of plasma membrane can be repaired within 5
s such that the egg can be fertilized and divide. This repair
process has an absolute requirement for Ca2 and is
mediated by recruitment of yolk granules which are lysosome-related organelles. Compelling evidence that a similar
repair process, recruiting conventional lysosomes, is a
feature of many mammalian cells has come from studies
by Andrews and colleagues (Reddy et al . 2001). They had
previously shown that manipulations of cultured NRK (normal
rat kidney) fibroblasts resulting in rises in cytosolic Ca2
concentration to the 1 /5 mM range could stimulate the
secretion of /10% of the total b -hexosaminidase content
of the cells (Rodriguez et al . 1997). This effect was mediated
by direct of lysosomes with the plasma membrane as
evidenced by the release of fluid phase tracers, increased
concentrations of lamps at the plasma membrane and EM
images of exocytic events. Fusion was dependent on the
integral membrane protein Ca2 sensor, synaptotagmin VII
(Martinez et al . 2000). More recently, Andrews and colleagues have shown that wounding-induced exocytosis of
lysosomes and plasma membrane resealing in NRK fibroblasts are dependent on both Ca2 and synaptotagmin VII
(Reddy et al . 2001). Interestingly, there is evidence that the
parasite Trypanosoma cruzi stimulates lysosomes to exocytose to create a site of entry into the cell (Andrews 2002).
Far from being simply terminal or dead-end organelles,
lysosomes can, thus, be regarded as very dynamic. They are
clearly capable of fusion with late endosomes with which they
are in equilibrium and are also able to fuse with the plasma
membrane under appropriate circumstances.
Membrane traffic into lysosomes
There are three well described routes for delivery of macromolecules to lysosomes: endocytosis, the biosynthetic route
(s) and autophagy (Figure 1).
Endocytosis
Whilst receptor mediated endocytosis via clathrin coated pits
is probably the best understood uptake route for extracellular
macromolecules to be delivered to lysosomes, it is certainly
not the only route. A variety of non-clathrin mediated
endocytic uptake pathways have been identified including
phagocytosis, caveolae-mediated uptake, macropinocytosis
and a constitutive cholesterol-sensitive non-clathrinmediated uptake pathway carrying lipid rafts to the Golgi
complex (all reviewed in Nichols and Lippincott-Scwartz
2001). Of these non-clathrin mediated uptake pathways,
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the most widely studied has been phagocytosis. Recent
exciting findings include the observation of fusion between
the endoplasmic reticulum and plasma membrane to generate the phagosome membrane in macrophages (Gagnon
et al . 2002) and molecular details of phagosome-lysosome
fusion from in vitro assays (Jahraus et al . 1998, 2001,
Peyron et al . 2001).
In the conventional endocytic pathway, there remain
disputes about whether membrane traffic from early-to-late
endosomes occurs via maturation (Murphy 1991, Stoorvogel
et al . 1991) or an endocytic carrier vesicle (ECV) formed as a
result of the recruitment to early endosomes of an isoform of
the COP-I coat responsible for retrograde traffic from the
Golgi complex to the ER (reviewed in Gu and Gruenberg
1999). Nevertheless, both the maturation and ECV models
provide for intermediates between early and late endosomes,
ensuring the morphological integrity of the latter compartment (Gruenberg and Maxfield 1995). As described above,
‘kiss and run’ events and direct fusion between late endosomes and lysosomes likely provide the major means of
delivery to lysosomes. The overall morphology of endosomal
compartments is, in major part, a consequence of the many
fusion events occurring in the endocytic pathway, in particular homotypic fusions of early endosomes, late endosomes
(Antonin et al . 2000) and lysosomes (Ward et al . 1997), as
well as heterotypic fusions between endocytic vesicles and
early endosomes, ECVs and late endosomes and late
endosomes and lysosomes. Very little or no fusion has
been observed between early endosomes and either late
endosomes or lysosomes (Aniento et al . 1993, Ward et al .
2000).
Biosynthetic routes
Most newly synthesized acid hydrolases are delivered to
lysosomes after being tagged with mannose 6-phosphate in
the cis-Golgi and binding to MPRs in the trans-Golgi network
(TGN). The bound hydrolases are first delivered to endosomes, where they dissociate from the receptors as a result
of the acidic lumenal pH, allowing the receptors to recycle to
the TGN. Fusion events between endosomes and preexisting lysosomes may be the major means of delivery
from endosomes to lysosomes (Luzio et al . 2000). Until
recently, it was thought that the MPRs were sorted into
clathrin coated pits at the TGN by means of the adaptor
protein AP-1. This adaptor is one of four: AP-1, AP-2 (used in
clathrin coated pits at the plasma membrane), AP-3 and AP4 (see below). All these adaptors are heterotetrameric
proteins utilised ubiquitously in post-Golgi secretory and
endocytic pathways. Electron microscopy, protein:protein
interaction studies and, most recently, structural biology
(Collins et al . 2002) have strongly suggested that adaptor
complexes have similar structures, resembling Mickey
Mouse, with a core or ‘head’ consisting of medium (m ) and
small sub-units and the amino-terminal domains of two large
sub-units (a /g and b ), flanked by flexibly-hinged ‘ears’
consisting of the carboxyterminal domains of the two large
sub-units. The results of experiments with m 1-deficient
mouse fibroblasts suggest that the major requirement for
AP-1 is in returning empty MPRs from endosomes to the
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TGN (Meyer et al . 2001). It now seems more likely that the
major clathrin adaptors required for traffic of MPRs and
bound hydrolases from the TGN to endosomes are the
GGAs (Golgi-localized, g -ear-containing, ADP ribosylation
factor-binding proteins) which are monomeric and only
distantly related to the heterotetrameric APs (Dell’Angelica
and Payne 2001, Puertollano et al . 2001, Robinson and
Bonifacino, 2001, Zhu et al . 2001). However, there are also
data showing the biochemical interaction of GGAs and AP-1
and demonstrating their colocalization in clathrin-coated
buds at the TGN. These data suggest that the role of
GGAs may be to package MPRs into AP-1-containing
clathrin coated vesicles for transport from the TGN to
endosomes (Doray et al . 2002).
Newly synthesized integral membrane proteins destined
for the limiting membrane of lysosomes are delivered either
by direct or indirect (via the plasma membrane) traffic routes
from the TGN to lysosomes. The evidence for individual
lamps taking mainly one or other of these routes has come
from kinetic studies of delivery and from the endocytic uptake
of anti-lamp antibodies or surface labelled lamps from the
plasma membrane. Thus, the type I integral membrane
proteins lamp-1 and lamp-2, which between them account
for over 50% of the lysosomal content of lamps (Andrejewski
et al . 1999), are delivered from the TGN with half times of
30 /90 min, mainly via an intracellular route (Barriocanal et
al . 1986, D’Souza and August 1986, Green et al. 1987,
Carlsson and Fukuda 1992, Akasaki et al . 1995, 1996). In
contrast, lysosomal acid phosphatase, also a type I membrane protein, is delivered with a half time of 5 /7 h, mainly
via the cell surface (Braun et al . 1989). Lamp-1, lamp-2 and
lysosomal acid phosphatase all have short, 10 /20 amino
acid, cytoplasmic tails containing tyrosine-based motifs that
determine their lysosomal targeting (Hunziker and Geuze
1996). The tyrosine-based signals are of the form YXX¥,
where X is any amino acid and ¥ is a bulky hydrophobic
amino acid, and are clearly related to internalization signals
of the same type, which interact with the m 2 sub-unit of the
AP-2 adaptor found in clathrin-coated pits at the cell surface
(Owen and Evans 1998, Bonifacino and Dell’Angelica 1999).
The specific residues used in the XX¥ positions can affect
the amount of direct vs indirect targeting to lysosomes
(Obermuller et al. 2002, Rous et al. 2002). In lamps, the
YXX¥ motif is often, but not always (Ihrke et al . 2000),
preceded by a G, which if mutated to A results in increased
trafficking via the cell surface. Not all lamps use tyrosinebased targeting signals for delivery to lysosomes. Cytoplasmic di-leucine-based motifs are also used, but their site of
interaction with adaptor sub-units is poorly understood.
There is also evidence that the lumenal and transmembrane
domains of some lamps also contain targeting information
(discussed in Reaves et al . 1998). Further complexity arises
in the case of polytopic lamps with several transmembrane
domains. In the case of the cystine transporter cystinosin,
which is predicted to contain seven transmembrane domains
and a 10 amino acid carboxyterminal cytoplasmic tail
containing the sequence GYDQL, lysosomal targeting is
dependent both on this tail and what appears to be a novel
targeting motif, containing the sequence YFPQA at its core,
within the third cytoplasmic loop (Cherqui et al . 2001).
The nature of the vesicular carriers used by lamps on the
direct route from the TGN to late endocytic compartments
has been difficult to establish. Whilst some early evidence
suggested that there may be some lamp-1 in AP-1 associated clathrin coated vesicles budding from the TGN
(Honing et al . 1996), the observation of neither a change
in steady state distribution nor mis-sorting to the cell surface
of lamp-1 in m lA-deficient cells suggested that AP-1 was
unlikely to play a major role in lysosomal targeting (Meyer et
al . 2000). GGAs also appear to play no role in lamp-1
delivery to lysosomes (Puertollano et al . 2001). Although a
function for AP-4 has been suggested in targeting lamps, this
adaptor is present in cells in low concentration and, to date,
there is no direct evidence of an involvement in the traffic of
any endogenous lamp (Aguilar et al. 2001). In contrast, there
is now compelling evidence that the adaptor protein most
important for the traffic of lamps from the TGN to lysosomes
by the direct route is AP-3. Mutations in AP-3 occur naturally
in animals including fruit flies and humans, leading to
alterations of eye colour in the former and a rare genetic
disease in the latter, as a result of defects in delivery of
proteins to lysosome-related organelles, i.e. Drosophila eye
pigment granules and platelet dense core granules, respectively (reviewed in Hirst and Robinson 1998, Huizing et al .
2001a, Mullins and Bonifacino 2001). Indeed, in Drosophila ,
eye colour mutants due to mutations in the genes encoding
each of the AP-3 sub-units have now been identified (d ,
garnet ; b 3, ruby ; m 3, carmine ; s 3, orange ). In mice, the
coat colour mutants pearl and mocha are, respectively,
caused by mutations in the genes encoding b 3B and d ,
resulting in abnormal membrane traffic to melanosomes.
Evidence that AP-3 is also responsible for targeting lamps to
conventional lysosomes originally came from experiments
where endogenous lamps were observed to traffic to lysosomes via the cell surface in cells in which functional AP-3
concentrations were reduced by transfecting with anti-sense
oligonucleotides to m 3A (Le Borgne et al . 1998) or in AP-3
deficient human cells (Dell’Angelica et al . 1999). More
recently, this result has been repeated in mouse pearl cells
with, in addition, the demonstration of ‘rescue’ after transfection and expression of b 3A or B (Peden et al . 2002). A
problem with these experiments has been that, whilst wild
type lamps are observed to traffic more via the cell surface in
AP-3 deficient cells, they can still reach lysosomes. Indeed, it
is difficult, if not impossible, to observe any deficiency of
lamp content in lysosomes of AP-3 deficient fibroblastic cells.
This is, presumably, because when the lamps reach the
plasma membrane they are efficiently internalized by clathrin-mediated endocytosis as a result of the interaction of
YXX¥ motifs with AP-2. Further evidence for the function of
AP-3 has come from the authors’ own experiments on lamp3 (CD63), which has four transmembrane domains and a
classical lysosomal targeting motif, GYEVM, at the end of a
short carboxyterminal cytoplasmic tail. Mutation of the
targeting motif to GYEVI produced a mutant lamp-3 tail
that, by yeast two hybrid analysis, was as good as the wild
type tail in binding to m 3, but no longer had any capability of
binding to m 2. When expressed in pearl or mocha mouse
cells, the lamp-3 mutant was trapped at the plasma
membrane and not delivered to lysosomes. In ‘rescued’
Lysosome biogenesis
mocha cells expressing the d sub-unit, transfected expressed GYEVI mutant of lamp-3 was delivered to lysosomes and not observed at the cell surface (Rous et al .
2002). Although it now seems clear that AP-3 is involved in
the traffic of membrane proteins to conventional lysosomes,
the intracellular site of action of AP-3 remains poorly defined.
It has been localized by microscopy at, or close to, the TGN
(Simpson et al . 1996) and on endosomes (Dell’Angelica et
al. 1998). Study of the trafficking of the AP-3 dependent
GYEVI mutant of lamp-3 suggests that it does not traffic via
the early endosomal subcompartment in which, during its
traffic itinerary, the TGN integral membrane protein TGN38 is
sorted for delivery to the TGN (Rous et al . 2002). Wherever
in the TGN/endosomal system AP-3 functions to deliver
lamps, fusion events between late endosomes and lysosomes will ensure delivery to the latter. An additional issue
with regard to the function of AP-3 is whether it requires
clathrin for vesicle formation. There have been reports of
partial colocalization of AP-3 and clathrin in cells, but the
clathrin binding site on b 3 is not required for its function
(Peden et al . 2002). The evidence that AP-3 is required for
the direct route to deliver lamps to lysosomes from the TGN
is consistent with the existence in yeast of an AP-3 trafficking
route from the late Golgi to the lysosome (Cowles et al .
1997, Piper et al . 1997, Stepp et al . 1997).
Autophagy
In a well-fed mammalian cell, endocytosis and biosynthetic
delivery are likely to be the major routes of membrane
delivery to lysosomes. However, upon nutrient starvation, the
process of autophagy (strictly, macroautophagy) is stimulated, resulting in the envelopment of a portion of cytoplasm
by membrane to form an autophagosome which is able to
fuse with lysosomes (Lawrence and Brown 1992, Yamamoto
et al . 1998). The origin of the autophagosome membrane
remains controversial, but recent genetic screens in yeast
have identified many genes which encode proteins essential
to the autophagic pathway (reviewed in Seaman and Luzio
2001, Noda et al. 2002). The study of these proteins and
their mammalian homologues is enabling rapid progress in
understanding the molecular mechanisms underlying autophagosome formation and consumption (Mizushima et al .
2001, Nara et al . 2002, Noda et al. 2002). In yeast, fusion of
autophagosomes with the vacuole requires many of the
same gene products as those required for delivering newly
synthesized vacuolar proteins (Darsow et al . 1997, Rieder
and Emr, 1997). By analogy, it is therefore, likely that, in
mammalian cells, the molecular mechanism of lysosome /
endosome fusion will be similar to that of lysosome /
autophagosome fusion.
Lysosome /endosome fusion and lysosome biogenesis
When proposing the ‘kiss and run’ hypothesis, Storrie and
Desjardins (1996) wrote that ‘when considering lysosome
biogenesis . . . the devil is in the details’, which they paraphrased as ‘the answer is in the mechanism’. The availability
of a cell-free system to study late endosome /lysosome
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fusion and the discovery of the hybrid organelle has opened
up the investigation of fusion events between late endosomes and lysosomes. Several clues have come from
studying the function of mammalian homologues of proteins
involved in delivery of macromolecules to vacuoles of the
yeast Saccharomyces cerevisiae , specifically those proteins
encoded by VPS (vacuolar protein sorting) and VAM
(vacuolar morphology) genes (Rothman and Stevens 1986,
Robinson et al . 1988, Bonangelino et al. 2002, Seeley et al .
2002). These genes have been discovered in a variety of
screens designed to isolate mutants with abnormal sorting of
newly synthesized proteins, in particular carboxypeptidase Y,
to the vacuole and/or abnormal vacuolar morphology. Newly
synthesized carboxypeptidase Y is targeted from the Golgi to
the vacuole as the result of the binding of a tetrapeptide
sequence within its pro-domain to a transmembrane receptor
Vps10p. For full activity, carboxypeptidase Y requires
proteolytic cleavage within the vacuole to produce the mature
form of the enzyme. Several of the VPS and VAM genes
were identified in more than one screen and have both VPS
and VAM designations, as well as PEP designations based
on reduced appearance of active carboxypeptidase Y in the
vacuole (Jones 1977). The vps mutants have been divided
into six classes (A /F) with respect to vacuolar morphology,
function and extent of protein sorting defects. Often, members of a class act on the same segment of the vacuolar
protein sorting pathway (reviewed in Seaman and Luzio
2001). Several late acting Vps/Vam proteins have been
shown to be required for homotypic fusion of yeast vacuoles,
a process that has also been reconstituted in a cell-free
system and is now well understood, with much molecular
detail available on priming, tethering, docking and fusion
steps (reviewed in Wickner 2002).
All of the membrane fusion events occurring throughout
the endocytic pathway are consistent with the action of the
common cytosolic fusion machinery, requiring NSF (Nethylmaleimide sensitive factor) and SNAPs (soluble NSF
attachment proteins), that functions according to the tenets
of the SNARE (SNAP receptor) hypothesis. This hypothesis,
proposed pairing between specific v- (vesicle) and t- (target
organelle) SNAREs, forms a functional trans-SNARE complex, which is at the centre of the docking and fusion of
vesicles with acceptor compartments (Rothman 1994).
Heterotypic fusion between late endosomes and lysosomes
to form hybrid organelles in the rat liver cell-free system is
ATP, cytosol and temperature dependent and requires the
presence of NSF, SNAPs and a small GTPase of the Rab
family (Mullock et al . 1998). The Rab requirement was
identified because fusion was inhibited by Rab-GDI (GDP
dissociation inhibitor). It has not been formally shown in the
mammalian cell-free system which Rab is required for late
endosome /lysosome fusion. The best candidate is Rab7,
the mammalian homologue of Ypt7p (Vam4p), which is
known to be required for yeast vacuole fusion (Wickner
2002), although Rab 14 has also been suggested (Harris and
Cardelli 2002). Fusion of late endosomes with lysosomes
can also be inhibited by the fast acting calcium chelator,
BAPTA (1,2-bis[2-aminophenoxy]ethane-N,N,N?,N?-tetraacetic acid) and is calmodulin dependent (Pryor et al . 2000a).
The availability of two inhibitors, Rab-GDI and BAPTA, has
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allowed the experimental ordering of the processes they
inhibit, with Rab-GDI acting very early in the fusion process
and BAPTA very late (Pryor et al. 2000a). By analogy with
what is known about homotypic yeast vacuole fusion where
Ypt7p is involved in recruiting putative tethering proteins and
there is a late calcium/calmodulin requirement for membrane
fusion, it has been possible to propose a model for the
mechanistic steps involved in late endosome /lysosome
fusion (Figure 3).
Tethering
There is a growing body of evidence that in many, if not all,
membrane fusion events on membrane traffic pathways,
specific tethering processes occur prior to trans-SNARE
complex formation, where tethering is defined as involving
links that extend over distances /25 nm from a given
membrane surface, in contrast to docking which holds
membranes within a bilayer’s distance, B/5 /10 nm of one
another (Pfeffer 1999). The physical existence of tethers late
in the endocytic pathway is not in dispute, since several
groups have observed fine striations, consistent with the
existence of tethers, between adjacent late endosomes and
lysosomes in morphological studies on cultured cells (van
Deurs et al . 1995, Futter et al . 1996, Bright et al. 1997).
Consistent with the model (Figure 3) in which the Rab acts
early and is involved in the recruitment of tethers are data
from experiments in which Rab7 was over-expressed in
mammalian cells and caused lysosome aggregation (Bucci
et al . 2000). Over-expression of dominant negative mutants
causes dispersion of lysosomes and they become inaccessible to endocytosed molecules. The presence of a Rab may
not be the only requirement early in the fusion process. The
phosphatidylinositol 3-kinase (Ptd-Ins 3-kinase) inhibitor
wortmannin also reduces fusion (Mullock et al . 1998).
Although a function for 3-phosphorylated phosphatidylinositides, including phosphatidylinositol 3-phosphate (Ptd-Ins 3phosphate), the product of Ptd-Ins 3-kinase activity, have not
been demonstrated in the fusion of late endosomes with
lysosomes, they are known to be involved in fusion events
elsewhere in the endocytic pathway. In particular, Ptd-Ins 3phosphate patches act synergistically with Rab5 in recruiting
tether proteins necessary for the homotypic fusion of early
endosomes (Simonsen et al . 1998). The best candidates for
proteins involved in the tethering complexes functioning late
in the endocytic pathway of mammalian cells are the
mammalian homologues of the yeast proteins Vps11p,
Vps16p, Vps18p, Vps33p, Vps39p and Vps41p (Seals et
al . 2000, Wurmser et al . 2000). In yeast, these proteins act
together as a complex (the HOPS, homotypic fusion and
vacuole protein sorting, or Class C Vps, complex) required
both for homotypic vacuole fusion and the docking of
vesicles delivering cargo on the biosynthetic pathway to the
vacuole (Sato et al . 2000). The complex binds to Ypt7 to
initiate docking and to a t-SNARE, Vam3p, enabling it to
regulate the formation of the functional trans-SNARE complex required for membrane fusion. There is some evidence
that a core Vps18p/Vps11p complex may function as a
tethering complex at multiple steps in membrane traffic
routes to the yeast vacuole, not simply at the final step of
delivery to the vacuole (Srivastava et al . 2000). Homologues
of the proteins making up the yeast HOPS complex exist in
both Drosophila and mammals. In both cases, they have
been implicated in the functioning of the late endocytic
pathway. Both VPS18 and VPS33 have Drosophila homologues, respectively deep orange and carnation , mutations
which affect the lysosome-related pigment granules in fly
eyes and, hence, eye colour (Sevrioukov et al . 1999). As in
yeast, they are associated in a large oligomeric complex.
Mammalian homologues have been cloned, shown by
Figure 3. The mechanism of late endosome /lysosome fusion. The diagram shows the possible stages of fusion of late endosomes with
lysosomes and reformation of lysosomes from the subsequent hybrid organelles. For simplicity, a single t-SNARE (Q-SNARE) is shown.
Tethering is shown preceding trans-SNARE assembly and fusion. Ca2 release from the organelle lumens (for simplicity, shown here only from
the endosome) is necessary for fusion. Reformation of lysosomes from the hybrid organelles should require a process to recycle the v-SNARE
(R-SNARE) for subsequent rounds of fusion and the condensation of lumenal content to produce dense core lysosomes.
Lysosome biogenesis
immunofluorescence microscopy to be associated with endocytic organelles and make up a large oligomeric complex
that interacts with the mammalian homologue of Vam3p,
syntaxin 7 (Huizing et al. 2001b, Kim et al . 2001; Figure 4).
Sequence analysis of the animal homologues has revealed
remarkable similarity in the domain structure amongst
Vps11p, Vpsl8p, Vam39p and Vps41p and thrown light on
the possible mechanism of oligomerization. All of these
proteins contain a clathrin heavy chain repeat (CLH) domain
that was originally analysed in the clathrin heavy chain and is
thought to promote the oligomerization of CLH-containing
proteins (Ybe et al. 1999). Mammalian Vps11p, Vpsl8p and
Vps41p also contain a complete or partial region that
encodes a RING-H2 domain. In both yeast and Drosophila ,
point mutations in key cysteine residues of the RING-H2
domain of Vpsl8p show that it is required for function
(Preston et al . 1991, Sevrioukov et al . 1999). Recent
evidence from over-expression of mammalian Vps39p shows
that it causes clustering of late endosomes and lysosomes
(Caplan et al . 2001). This effect was observed in the
presence of dominant negative Rab7, implying that it functions downstream of, or in parallel to, Rab7.
Trans-SNARE complex formation
Syntaxin 7 has been firmly implicated as a SNARE required
for both heterotypic fusion of late endosomes with lysosomes
and homotypic fusion of late endosomes. Using cell-free
systems, antibodies to this SNARE have been shown to
inhibit both of these fusion events (Antonin et al . 2000,
Mullock et al . 2000, Ward et al . 2000). In addition, overexpression of the cytoplasmic domain inhibits delivery of
endocytosed markers to late endocytic compartments (Nakamura et al . 2000). Whilst it is reassuring that there are now
data linking this SNARE to a possible tethering complex,
described above, an unresolved question is how syntaxin 7
can apparently mediate both homotypic and heterotypic
Figure 4. Possible proteins involved in tethering late endosomes
and lysosomes prior to membrane fusion. Likely proteins include the
mammalian homologues of yeast Vps11p, Vps 16p, Vps18p,
Vps33p, Vps39p and Vps41p which form a large oligomeric complex
interacting with syntaxin 7. Rab7 is the most likely Rab involved. In
the diagram, partly based on the analogy with the yeast vacuole
system (Sato et al . 2000), syntaxin 7 is shown as a t-SNARE on the
lysosome. It is also present on late endosomes, which would also be
able to recruit the tethering complex.
147
fusion processes involving late endosomes. One possibility
is that it has different, combinatorial partners to form transSNARE complexes separately mediating the two events.
Although the SNARE hypothesis proposed specific pairing
between v-(vesicle) and t-(target organelle) SNAREs, an
alternative classification based on sequence alignments of
the coiled coil domains and structural features observed in
the crystal structure of the heterotrimeric synaptic fusion
complex (Sutton et al . 1998) has been proposed. This
separates SNAREs into Q-SNAREs and R-SNAREs, with
four-helix bundles composed of 3 Q-SNARE and 1 R-SNARE
helices being formed after SNARE complex assembly
(Fasshauer et al . 1998). A refinement of this classification
separates the Q-SNAREs into Qa, Qb and Qc, requiring one
of each and an R-SNARE to form the functional SNARE
complex (Bock et al. 2001). An alternative nomenclature for
SNAREs has been proposed whereby the t-SNARE consists
of a heavy chain and two light chains and the v-SNARE a
single polypeptide chain (Fukuda et al . 2000). In practice, for
a given SNARE complex, the v-SNARE is synonymous with
the R-SNARE and the t-SNARE with the three Q-SNAREs. A
refinement occurs, sometimes, when a vesicle fuses with the
plasma membrane, needing only 3 SNARES, one of which
contains two Q helices or, in the alternative nomenclature, a
combined light chain with two helices.
For homotypic fusion of late endosomes, antibody inhibition data has shown that the partners of syntaxin 7 in the
trans-SNARE complex are syntaxin 8, Vti1b, and Vamp 8
(Antonin et al . 2000). These four SNAREs provide, respectively, the necessary Qa, Qb, Qc and R SNAREs for a
functional complex. The core of this complex has been
crystallized and its structure solved (Antonin et al . 2002a).
Less is known about the trans-SNARE complex for heterotypic fusion of late endosomes and lysosomes. In experiments using membrane fractions of melanoma cells, which
express much more syntaxin 7 than other cell types
investigated, it was found that immunoprecipitation of syntaxin 7 co-precipitated Vamp 7 and syntaxin 6 along with
Vti1b and Vamp 8 (Wade et al . 2001). There is no functional
evidence indicating a role for syntaxin 6 late in the endocytic
pathway, nor evidence of a function for Vamp 8 in heterotypic
late endosome /lysosome fusion (Mullock et al . 2000). In
contrast, there are data showing inhibition of late endosome /lysosome fusion by a bacterially expressed Vamp
7 lacking its transmembrane domain (Ward et al . 2000),
raising the intriguing possibility that this R-SNARE can form
an alternative trans-SNARE complex with syntaxin 7 for late
endosome /lysosome fusion. Consistent with this suggestion, a SNARE complex consisting of orthologues of mammalian syntaxin 7, syntaxin 8, Vti1 and Vamp 7 has been
isolated from Dictyostelium discoideum (Bogdanovic et al .
2002). Permeabilized mammalian cell assays, in which
antibodies to specific SNAREs have been found to inhibit
degradation of endocytosed epidermal growth factor, have
also implicated Vamp 7 in the pathway from early endosomes to lysosomes (Advani et al . 1999). Vamp 7 (also
known as toxin insensitive- or TI-Vamp or VampSYBL1) is an
unusual R-SNARE in having a relatively long ( /110 amino
acid) regulatory domain at its aminoterminus (Martinez-Arca
et al . 2000, Filippini et al . 2001), which is predicted to have a
148
J.P. Luzio et al.
structural similarity to the s sub-unit of AP-2 (Collins et al .
2002) and may well have a role in regulating fusion events. In
addition, an helical aminoterminal domain in syntaxin 7 has
been implicated in the regulation of SNARE complex
assembly (Antonin et al . 2002b). Amongst the tethering
complex proteins functioning late in the endocytic pathway,
Vps33p, which is a Sec1p homologue, is also predicted to act
as a negative regulator of SNARE complex assembly. Thus,
despite the lack of certainty with regard to the composition of
the trans-SNARE complex required for heterotypic late
endosome /lysosome fusion, there are already several clues
as to how assembly of this complex may be regulated.
Membrane fusion: the role of calcium
In homotypic yeast vacuole fusion, calcium is released from
the lumen of the docked organelles after trans-SNARE
complex formation and mediates fusion via calmodulin
dependent event(s) (Peters and Mayer 1998). The calcium/
calmodulin dependence has been proposed to be mediated
via a protein complex which includes a protein phosphatase
(Peters et al . 1999) and/or a direct interaction with V0, the
membrane-integral sector of the vacuolar H -ATPase (Peters et al . 2001). Peters et al . (2001) have proposed the
formation of V0 trans-complexes, acting as proteolipid-lined
channels at the fusion site. Additional protein factors, the Vtc
or vacuolar transporter chaperones, are required for this
process (Müller et al . 2002). Key experiments carried out by
Peters and Mayer (1998) showed that the fast acting Ca2
chelator BAPTA, the Ca2 ionophore ionomycin and Ca 2ATPase inhibitors all blocked homotypic vacuole fusion.
They were also able to deplete lumenal Ca2 stores by
treatment with BAPTA in the presence of ionomycin and
showed that such Ca2 depleted vacuoles were unable to
fuse. When lumenal Ca2 was restored, the vacuoles again
became fusion competent.
Using cell-free systems, established with sub-cellular
fractions from mammalian cells, complementary data have
been obtained for homotypic fusion of early endosomes
(Holroyd et al. 1999) and heterotypic fusion of late endosomes with lysosomes (Pryor et al . 2000a). In both systems,
fusion was inhibited by BAPTA and was also prevented by
pre-incubation with a membrane permeable methyl ester of
EGTA, EGTA-AM, which is cleaved within the organelle
lumen resulting in chelation of lumenal Ca2 . The inhibitory
effects of BAPTA and EGTA-AM were reversed by addition
of CaCl2. These data strongly support a role for the release
of Ca2 from the endosome lumen in fusion events in the
endocytic pathway. Experiments in which increasing concentrations of CaCl2 were added to fusion assays in the
presence of BAPTA suggested that 0.3 mM Ca2 is optimal
for early endosome fusion (Holroyd et al . 1999) and 0.5 mM
Ca2 for fusion of late endosomes with lysosomes (Pryor et
al . 2000a). As shown in Figure 2, there is also evidence in
the latter system that the BAPTA effect occurs later in the
docking/fusion process than inhibition by Rab-GDI, consistent with the observation in yeast vacuole fusion that Ca2
release occurs after trans-SNARE complex formation. Release of lumenal Ca2 is likely to be important in fusion
events other than those occuring in the endocytic pathway
(Pryor et al. 2000b). Lumenal Ca2 has been implicated in
the fusion of nuclear membranes (Sullivan et al . 1993), of
regulated secretory granules required to sustain optimal
exocytosis (Scheenen et al . 1998) and for vesicular traffic
through the Golgi complex (Porat and Elazar, 2000).
Consistent with a requirement for release of lumenal Ca2
to mediate lysosome-late endosome fusion, there are recent
data reporting the Ca2 concentration in lysosomes as being
in the 400 /600 mM range, i.e. very much higher than
cytosolic concentrations (Christensen et al . 2001). This
high lysosomal concentration of Ca2 seems at odds with
a study showing that endocytosed Ca2 is rapidly lost from
endosomal compartments of fibroblasts with time, such that
the endosomal concentration of Ca2 was only 3 mM, 20 min
after uptake from extracellular medium containing 2 mM
Ca2 . However, there is also evidence that both endosomal
(Hilden and Madias 1989) and lysosomal (Lemons and
Thoene 1991) membranes contain Ca2 transport systems,
suggesting that the high intra-lysosomal Ca2 concentration
may be the result of transport across the lysosomal
membrane rather than endocytosis from the extracellular
medium.
The mechanism by which Ca2 released from the lumen
of mammalian endocytic compartments mediates membrane
fusion is much less well understood than in yeast. The effect
of calmodulin antagonists to inhibit cell-free late endosome /
lysosome fusion is consistent with a calmodulin-mediated
mechanism as in yeast vacuole fusion. However, other
possibilities include interaction of Ca2 with the SNARE
machinery or with annexins (reviewed in Pryor et al . 2000b)
or an effect on a synaptotagmin isoform. The latter is
particularly intriguing, because of data showing that synaptotagmin VII is localized to lysosomes and plays a key role in
Ca2 stimulated fusion of lysosomes with the plasma
membrane (see above). However, in several cell types,
fusion of lysosomes with the plasma membrane is triggered
when the cytosolic Ca2 concentration rises to /1 /5 mM
(Rodriguez et al . 1997, Martinez et al . 2000), /3 /15-fold
higher than that found to be optimal for fusion of late
endosomes with lysosomes (Pryor et al . 2000a). Indeed,
Ca2 concentrations above 1 mM inhibit heterotypic fusion of
late endosomes with lysosomes (Pryor et al . 2000a).
Although a wide variety of calmodulin activated enzymes,
transporters, cytoskeletal proteins and other proteins are
known (reviewed in Pryor et al . 2000b), no downstream
calmodulin effector for Ca2 -dependent fusion events in the
mammalian endocytic pathway has been described. The
requirement for a downstream effector of calmodulin for cellfree late endosome /lysosome fusion is consistent with the
observed displacement of the time course of BAPTA inhibition of this process from the time course when stopping the
reaction by placing the assay tubes on ice (Pryor et al .
2000a).
If lumenal Ca2 is essential for efficient membrane traffic
in the endocytic pathway, one prediction is that diseases
which result in an alteration of lumenal Ca2 concentration
may show defects in the endocytic pathway. In this context, it
is interesting to note the recent cloning of the gene causing
mucolipidosis type IV (Bargal et al . 2000, Bassi et al . 2000,
Sun et al . 2000). This disease is characterized by abnormal
Lysosome biogenesis
late endosomal/lysosomal structures which are filled with
multilamellar membranous whorls and do not show the
characteristic morphology of dense core lysosomes by
electron microscopy. There are also defects in sorting and/
or transport in the late endocytic pathway (Chen et al . 1998).
The mucolipidosis IV gene encodes mucolipin-1 which is
predicted to be a lysosomal integral membrane protein with
several trans-membrane domains. Mucolipin-1 has sequence similarities to members of the TRP (transient
receptor potential) superfamily, in particular the polycystins,
and has been shown to be a novel Ca2-permeable channel
modulated by changes in Ca2 concentration (LaPlante et
al . 2002). There is evidence that CUP-5, the Caenorhabditis
elegans homologue of mucolipin-1, functions in the biogenesis of lysosomes, possibly in the process of reformation
from hybrid organelles (Fares and Grant 2002).
Membrane traffic out of lysosomes
After fusion of lysosomes with late endosomes, lysosomes
are reformed from the resultant hybrid organelles. This
process requires condensation of content, since lysosomes
are denser than hybrid organelles. It also requires the
removal of membrane proteins that need to be recycled
and/or are found in late endosomes but not lysosomes.
Condensation of lumenal content appears to occur by a
process similar to that occurring during the formation of
mature regulated secretory granules in neuroendocrine cells
(Bauerfeind and Huttner 1993, Tooze 1998, Arvan et al .
2002). Just as for regulated secretory granules, there is
evidence that lumenal Ca2 and low pH are required for
formation of dense core lysosomes, since treatment of hybrid
organelles with either EGTA-AM or bafilomycin A1, an
inhibitor of the vacuolar proton pumping ATPase, can
prevent this happening in a cell-free system (Pryor et al .
2000a).
Of membrane proteins that need to be removed from the
hybrid organelle or recycled during the reformation of
lysosomes, the most obvious are the MPRs, since by
definition these are not found in lysosomes. The process
whereby they are recycled to the TGN has been studied in
several laboratories and appears mostly to occur from
endosomes, such that late endosomes which fuse with
lysosomes are already relatively depleted in MPRs (Hirst et
al . 1998). Two proteins required for recycling of MPRs have
been identified as Rab 9 and TIP47 (tail interacting protein of
47kDa). TIP47 binds to separate determinants in the
cytoplasmic tails of the two MPRs and is likely to be involved
in cargo selection into the recycling vesicles (Diaz and
Pfeffer 1998). TIP47 also binds directly to Rab9 (Hanna et
al . 2002). The nature of the coat required for the recycling
vesicles is not known. As described above, AP-1 has been
suggested as being involved in MPR recycling. However, it
now seems clear that there is more than one recycling
pathway from endocytic compartments to the TGN, which
may differ both in adaptor molecules and coats (Pfeffer
2001). In yeast, the protein components of the vesicle coat
required for recycling of Vps10p, the carboxypeptidase Y
receptor, have been identified. These proteins are Vps5p,
149
Vpsl7p, Vps26p, Vps29p and Vps35p, which together form
the retromer coat complex (Seaman et al . 1998, Reddy and
Seaman 2001). Homologues of each of these retromer
components have been identified in mammalian cells,
suggesting that this coat is universally important in retrograde traffic from endocytic compartments to the TGN (Haft
et al . 2000, Pfeffer 2001). To date, there is no published
evidence as to what cargo retromer coated vesicles may
contain in mammalian cells.
The MPR recycling vesicles leaving late endosomes and
hybrid organelles may not remove all other membrane
proteins necessary for reformation of lysosomes and/or
lysosome biogenesis. The Niemann-Pick C protein (NPC1),
which is an integral membrane protein with 13 predicted
transmembrane domains, is thought to be required for
vesicular shuttling of cholesterol, derived from hydrolysed
low density lipoprotein, from late endocytic organelles to the
trans-Golgi network (Liscum 2000). There is evidence of
colocalization of some of the R-SNARE, Vamp 7, in the
shuttling vesicles, raising the possibility that these vesicles
may play a role in recycling SNARE components required for
fusion events late in the endocytic pathway as well as
removing cholesterol, a key product of lysosomal hydrolysis
(Ko et al . 2001).
Although clathrin is able to form coated vesicles on late
endocytic organelles (Traub et al. 1996), it is unlikely to be
involved in lysosome reformation from hybrid organelles,
since cells engineered to have no clathrin have normal dense
core lysosomes (Wettey et al . 2002).
Lumenal membranes in lysosomes
A widely recognized morphological feature of late endocytic
organelles, including lysosomes, is the presence of lumenal
membrane. Indeed, the term multivesicular body is often
used instead of late endosome (Piper and Luzio 2001).
Whilst some of the lumenal membrane may derive as a byproduct of fusion events, resulting from the release of
apposed membrane into the lumen of a fused organelle
(Wang et al . 2002), this is not the source of the majority. The
lumenal membrane, when compared with the limiting membrane, is enriched in lysobisphosphatidic acid (LBPA) or PtdIns 3-phosphate, the latter being in distinct regions low in
LBPA (Gillooly et al . 2000). In some cells, there is good
evidence for a dynamic relationship between the lumenal and
limiting membrane, such that the former can fuse with the
latter, greatly increasing the surface area of the organelle
(Kleijmeer et al . 2001). The lumenal membrane may be
secreted as a result of fusion of late endocytic organelles
with the cell surface, being released as exosomes which are
known to function in intercellular communication during the
immune response (Stoorvogel et al . 2002, Thery et al .
2002).
It is now recognized that the sorting of membrane proteins
into lumenal membrane in the endocytic pathway is a key
event in targeting these proteins for lysosomal degradation.
In many cases, both in yeast and mammalian cells, an
ubiquitin tag is the trigger for lumenal sorting, but some
proteins enter the lumenal vesicles in an ubiquitin-indepen-
150
J.P. Luzio et al.
dent manner (Reggiori and Pelham 2001). Studies in yeast
have identified much of the cargo selection machinery for
ubiquitin-dependent sorting of proteins to be degraded into
the lumenal membranes of endosomes (Conibear 2002).
Three separate complexes of class E Vps proteins, known as
ESCRT (endosomal sorting complexes required for transport) complexes I, II and III, acting sequentially, appear to
play a major role (Katzmann et al . 2001, Babst et al .
2002a,b). ESCRT-I binds directly to ubiquitinated cargo, and
mutation of the ubiquitin binding domain of the ESCRT-I
component Vps23p prevents sorting of cargo into lumenal
vesicles and also formation of the vesicles, indicating a
coupling of the machinery for sorting and lumenal vesicle
formation. ESCRT-III recruits a deubiquitinating enzyme
Doa4p and, in a final step, the AAA-ATPase Vps4p catalyses
the release of the machinery from the membrane. A further
Class E Vps protein, Vps27p, in complex with Hse1p, also
binds to ubiquitinated membrane proteins and acts as a
sorting receptor for degradation (Bilodeau et al . 2002). It
may act upstream of ESCRT-I or independently.
In several cases, orthologues of individual components of
this yeast sorting machinery have been shown to play an
equivalent role in mammalian cells. For example, mutations
in Tsg101, the human orthologue of Vps23p, affect traffic of
the epidermal growth factor receptor, which is normally
delivered to endosomal lumenal vesicles as a necessary
pre-requisite to its efficient degradation late in the endocytic
pathway (Babst et al. 2000). Similarly, the mammalian
Vps27p orthologue Hrs also binds ubiquitinated proteins
and is able to target these for lysosomal degradation
(Raiborg et al . 2002). Intriguingly, Hrs sorts ubiquitinated
membrane proteins into clathrin-coated microdomains on the
early endosome. EM studies have shown that these clathrin
coats are devoid of the adaptors AP-1, AP-2 and AP-3 and
do not cause budding off of coated vesicles (Sachse et al .
2002). They do appear to concentrate membrane proteins
destined for delivery to lumenal vesicles and degradation,
together with proteins such as syntaxin 7, which remains on
the limiting membrane and is required for fusion events later
in the endocytic pathway.
In mammalian cells, it is clear that the formation of lumenal
membrane vesicles in the endocytic pathway commences at
the early endosome. This internal membrane is delivered to
late endosmes and, eventually, after fusion with lysosomes
to the proteolytic environment of the hybrid organelle. The
lumenal membrane observed in dense core lysosomes may,
at least in part, constitute remnants of the endosomal
lumenal vesicles delivered to the hybrid.
Conclusions
Recent advances in understanding of the molecular biology
of membrane traffic pathways have contributed greatly to
understanding of lysosome dynamics. The challenge now is
to understand better the oligomeric protein complexes
functioning in lysosome biogenesis, sorting and fusion
events. This will provide insights to specificity in lysosome
function, e.g. what are the differences in machinery required
for fusion with late endosomes vs the plasma membrane. In
turn, this should lead to an understanding of the regulation of
lysosome function and, eventually, a better knowledge of
abnormalities associated with disease processes involving
lysosomes.
Acknowledgements
Experimental work in our laboratories is funded by the Medical
Research Council, Wellcome Trust and National Institutes of Health
(RCP). We thank our colleagues, especially Nick Bright and Matthew
Seaman, for much valuable discussion.
References
Advani, R. J., Yang, B., Prekeris, R., Lee, K. C., Klumperman, J. and
Scheller, R. H., 1999, VAMP-7 mediates vesicular transport from
endosomes to lysosomes. Journal of Cell Biology , 146, 765 /776.
Aguilar, R. C., Boehm, M., Gorshkova, I., Crouch, R. J., Tomita, K.,
Saito, T., Ohno, H. and Bonifacino, J. S., 2001, Signal-binding
specificity of the m4 subunit of the adaptor protein complex AP-4.
Journal of Biological Chemistry , 276, 13145 /13152.
Akasaki, K., Michihara, A., Fujiwara, Y., Mibuka, K. and Tsuji, H.,
1996, Biosynthetic transport of a major lysosome-associated
membrane glycoprotein 2, lamp-2: a significant fraction of newly
synthesized lamp-2 is delivered to lysosomes by way of early
endosomes. Journal of Biochemistry , 120, 1088 /1094.
Akasaki, K., Michihara, A., Mibuka, K., Fujiwara, Y. and Tsuji, H.,
1995, Biosynthetic transport of a major lysosomal membrane
glycoprotein, lamp-1: convergence of biosynthetic and endocytic
pathways occurs at three distinctive points. Experimental Cell
Research , 220, 464 /473.
Andrews, N. W., 2000, Regulated secretion of conventional lysosomes. Trends in Cell Biology , 10, 316 /321.
Andrews, N. W., 2002, Lysosomes and the plasma membrane:
trypanosomes reveal a secret relationship. Journal of Cell
Biology , 158, 389 /394.
Andrejewski, N., Punnonen, E.-L., Guhde, G., Tanaka, Y., LüllmannRauch, R., Hartmann, D., von Figura, K. and Saftig, P., 1999,
Normal lysosomal morphology and function in LAMP-1-deficient
mice. Journal of Biological Chemistry , 274, 12692 /12701.
Aniento, F., Emans, N., Griffiths, G. and Gruenberg, J., 1993,
Cytoplasmic dyneindependent vesicular transport from early to
late endosomes. Journal of Cell Biology , 123, 1373 /1387.
Antonin, W., Dulubova, I., Arac, D., Pabst, S., Plitzner, J., Rizo, J.
and Jahn R., 2002b, The N-terminal domains of syntaxin 7 and vti
1b form three-helix bundles that differ in their ability to regulate
SNARE complex assembly. Journal of Biological Chemistry, in
press.
Antonin, W., Fasshauer, D., Becker, S., Jahn, R. and Schneider, T.
R., 2002, Crystal structure of the endosomal SNARE complex
reveals common structural principles of all SNAREs. Nature
Structure Biology , 9, 107 /111.
Antonin, W., Holroyd, C., Fasshauer, D., Pabst, S., Von Mollard, G.
F. and Jahn, R., 2000, A SNARE complex mediating fusion of late
endosomes defines conserved properties of SNARE structure
and function. EMBO Journal , 19, 6453 /6464.
Arvan, P., Zhang, B.-Y., Feng, L., Liu, M. and Kuliawat, R., 2002,
Lumenal protein multimerization in the distal secretory pathway/
secretory granules. Current Opinions in Cell Biology , 14, 448 /
453.
Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T. and
Emr, S. D., 2002, ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting. Developmental Cell , 3, 271 /282.
Babst, M., Katzmann, D. J., Snyder, W. B., Wendland, B. and Emr,
S. D., 2002, Endosome-associated complex, ESCRT-II, recruits
transport machinery for protein sorting at the multivesicular body.
Developmental Cell , 3, 283 /289.
Babst, M., Odorizzi, G., Estepa, E. J. and Emr, S. D., 2000,
Mammalian tumor susceptibility gene 101 (TSG101) and the
Lysosome biogenesis
yeast homologue, Vps23p, both function in late endosomal
trafficking. Traffic , 1, 248 /258.
Bainton, D. F., 1981, The discovery of lysosomes. Journal of Cell
Biology , 91, 66s /76s.
Bargal, R., Avidan, N., Ben-Asher, E., Olender, Z., Zeigler, M.,
Frumkin, A., Raas-Rothschild, A., Glusman, G., Lancet, D. and
Bach, G., 2000, Identification of the gene causing mucolipidosis
type IV. Nature Genetics , 26, 120 /123.
Barois, N., de Saint-Vis, B., Lebecque, S., Geuze, H. J. and
Kleijmeer, M. J., 2002, MHC class II compartments in human
dendritic cells undergo profound structural changes upon activation. Traffic , 3, 894 /905.
Barriocanal, J. G., Bonifacino, J. S., Yuan, L. and Sandoval, I. V.,
1986, Biosynthesis, glycosylation, movement through the Golgi
system, and transport to lysosomes by an N-linked carbohydrateindependent mechanism of three lysosomal integral membrane
proteins. Journal of Biological Chemistry , 261, 16755 /16763.
Bassi, M. T., Manzoni, M., Monti, E., Pizzo, M. T., Ballabio, A. and
Borsani, G., 2000, Cloning of the gene encoding a novel integral
membrane protein, mucolipidin- and identification of the two major
founder mutations causing mucolipidosis type IV. American
Journal of Human Genetics , 67, 1110 /1120.
Bauerfeind, R. and Huttner, W. B., 1993, Biogenesis of constitutive
secretory vesicles, secretory granules and synaptic vesicles.
Current Opinion in Cell Biology , 5, 628 /635.
Bilodeau, P. S., Urbanowski, J. L., Winistorfer, S. C. and Piper, R. C.,
2002, The Vps27p Hse1p complex binds ubiquitin and mediates
endosomal protein sorting. Nature Cell Biology , 4, 534 /539.
Blott, E. J. and Griffiths, G. M., 2002, Secretory lysosomes. Nature
Reviews in Molecular Cell Biology , 3, 122 /131.
Bock, J. B., Matern, H. T., Peden, A. A. and Scheller, R. H., 2001, A
genomic perspective on membrane compartment organization.
Nature , 409, 839 /841.
Bogdanovic, A., Bennett, N., Kieffer, S., Louwagie, M., Morio, T.,
Garin, J., Satre, M. and Bruckert, F., 2002, Syntaxin 7, Syntaxin
8, Vti1 and VAMP7 form an active SNARE complex for early
macropinocytic compartment fusion in Dictyostelium discoideum .
Biochemical Journal , 368, 29 /39.
Bonangelino, C. J., Chavez, E. M. and Bonifacino, J. S., 2002,
Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae . Molecular Biology of the Cell , 13, 2486 /
2501.
Bonifacino, J. S. and Dell’Angelica, E. C., 1999, Molecular bases for
the recognition of tyrosine-based sorting signals. Journal of Cell
Biology , 145, 923 /926.
Braun, M., Waheed, A. and von Figura, K., 1989, Lysosomal acid
phosphatase is transported to lysosomes via the cell surface.
EMBO Journal , 8, 3633 /3640.
Bright, N. A., Reaves, B. J., Mullock, B. M. and Luzio, J. P., 1997,
Dense core lysosomes can fuse with late endosomes and are reformed from the resultant hybrid organelles. Journal of Cell
Science , 110, 2027 /2040.
Bucci, C., Thomsen, P., Nicoziani, P., McCarthy, J. and van Deurs,
B., 2000, Rab7: a key to lysosome biogenesis. Molecular Biology
of the Cell , 11, 467 /480.
Caplan, S., Hartnell, L. M., Aguilar, R. C., Naslavsky, N. and
Bonifacino, J. S., 2001, Human Vam6p promotes lysosome
clustering and fusion in vivo. Journal of Cell Biology , 154, 109 /
122.
Carlsson, S. R. and Fukuda, M., 1992, The lysosomal membrane
glycoprotein lamp-1 is transported to lysosomes by two alternative pathways. Archives of Biochemistry and Biophysics , 296,
630 /639.
Casciola-Rosen, L. A. and Hubbard, A. L., 1991, Hydrolases in
intracellular compartments of rat liver cells. Evidence for selective
activation and/or delivery. Journal of Biological Chemistry , 266,
4341 /4347.
Chen, C.-S., Bach, G. and Pagano, R. E., 1998, Abnormal transport
along the lysosomal pathway in Mucolipidosis type IV disease.
Proceeding of the National Academy of Sciences (USA) , 95,
6373 /6378.
Cherqui, S., Kalatzis, V., Trugnan, G. and Antignac, C., 2001, The
targeting of cystinosin to the lysosomal membrane requires a
151
tyrosine-based signal and a novel sorting motif. Journal of
Biological Chemistry , 276, 13314 /13321.
Chow, A., Toomre, D., Garrett, W. and Mellman, I., 2002, Dendritic
cell maturation triggers retrograde MHC class II transport from
lysosomes to the plasma membrane. Nature , 418, 988 /994.
Christensen, K. A., Myers, J. T. and Swanson, J. A., 2001, pHdependent regulation of lysosomal calcium in macrophages.
Journal of Cell Science , 115, 599 /607.
Collins, B. M., McCoy, A. J., Kent, H. M., Evans, P. R. and Owen, D.
J., 2002, Molecular architecture and functional model of the
endocytic AP2 complex. Cell , 109, 523 /535.
Conibear, E., 2002, An ESCRT into the endosome. Molecular Cell ,
10, 215.
Cowles, C. R., Odorizzi, G., Payne, G. S. and Emr, S. D., 1997, The
AP-3 adaptor complex is essential for cargo-selective transport to
the yeast vacuole. Cell , 91, 109 /118.
Darsow, T., Rieder, S. E. and Emr, S. D., 1997, A multispecificity
syntaxin homologue, Vam3p, essential for autophagic and
biosynthetic protein transport to the vacuole. Journal of Cell
Biology , 138, 517 /529.
de Duve, C., 1983, Lysosomes revisited. European Journal of
Biochemistry , 137, 391 /397.
Dell’Angelica, E. C. and Payne, G. S., 2001, Intracellular cycling of
lysosomal enzyme receptors: cytoplasmic tails’ tales. Cell , 106,
395 /398.
Dell’Angelica, E. C., Klumperman, J., Stoorvogel, W. and Bonifacino,
J. S., 1998, Association of the AP-3 adaptor complex with clathrin.
Science , 280, 431 /434.
Dell’Angelica, E. C., Shotelersuk, V., Aguilar, R. C., Gahl, W. A. and
Bonifacino, J. S., 1999, Altered trafficking of lysosomal proteins in
Hermansky-Pudlak syndrome due to mutations in the b3A subunit
of the AP-3 adaptor. Molecular Cell , 3, 11 /21.
Diaz, E. and Pfeffer, S. R., 1998, TIP47: a cargo selection device for
mannose 6 phosphate receptor trafficking. Cell , 93, 433 /443.
Doray, B., Ghosh, P., Griffith, J., Geuze, H. J. and Kornfeld, S., 2002,
Cooperation of GGAs and AP-1 in packaging MPRs at the transGolgi network. Science , 297, 1700 /1703.
D’Souza, M. P. and August, J. T., 1986, A kinetic analysis of
biosynthesis and localization of a lysosome-associated membrane glycoprotein. Archives of Biochemistry and Biophysics ,
249, 522 /532.
Fares, H. and Grant, B., 2002, Deciphering endocytosis in Caenorhabditis elegans. Traffic , 3, 11 /19.
Fasshauer, D., Sutton, R. B., Brunger, A. T. and Jahn, R., 1998,
Conserved structural features of the synaptic fusion complex:
SNARE proteins reclassified as Q- and R-SNAREs. Proceedings
of the National Academy of Sciences (USA) , 95, 15781 /15786.
Filippini, F., Rossi, V., Galli, T., Budillon, A., D’Urso, M. and
D’Esposito, M., 2001, Longins: a new evolutionary conserved
VAMP family sharing a novel SNARE domain. Trends in
Biochemical Science , 26, 407 /409.
Fukuda, R., McNew, J. A., Weber, T., Parlati, F., Engel, T., Nickel,
W., Rothman, J. E. and Sollner, T. H., 2000, Functional
architecture of an intracellular membrane t-SNARE. Nature ,
407, 198 /202.
Futter, C. E., Pearse, A., Hewlett, L. J. and Hopkins, C. R., 1996,
Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes.
Journal of Cell Biology , 132, 1011 /1023.
Gagnon, E., Duclos, S., Rondeau, C., Chevet, E., Cameron, P. H.,
Steele-Mortimer, O., Paiement, J., Bergeron, J. J. M. and
Desjardins, M., 2002, Endoplasmic reticulum mediated phagocytosis is a mechanism of entry into macrophages. Cell , 110, 119 /
131.
Gillooly, D. J., Morrow, I. C., Lindsay, M., Gould, R., Bryant, N. J.,
Gaullier, J. M., Parton, R. G. and Stenmark, H., 2000, Localization
of phosphatidylinositol 3-phosphate in yeast and mammalian
cells. EMBO Journal , 19, 4577 /4588.
Green, S. A., Zimmer, K. P., Griffiths, G. and Mellman, I., 1987,
Kinetics of intracellular transport and sorting of lysosomal
membrane and plasma membrane proteins. Journal of Cell
Biology , 105, 1227 /1240.
152
J.P. Luzio et al.
Griffiths, G., 1996, On vesicles and membrane compartments.
Protoplasma , 195, 37 /58.
Gruenberg, J. and Maxfield, F. R., 1995, Membrane transport in the
endocytic pathway. Current Opinion in Cell Biology , 7, 552 /563.
Gu, F. and Gruenberg, J., 1999, Biogenesis of transport intermediates in the endocytic pathway. FEBS Letters , 452, 61 /66.
Haft, C. R., de la Luz Sierra, M., Bafford, R., Lesniak, M. A., Barr, V.
A. and Taylor, S. I., 2000, Human orthologs of yeast vacuolar
protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes. Molecular Biology of the Cell , 11, 4105 /4116.
Hanna, J., Carroll, K. and Pfeffer, S. R., 2002, Identification of
residues in TIP47 essential for Rab9 binding. Proceedings of the
National Academy of Sciences (USA) , 99, 7450 /7454.
Harding, C. V., Collins, D. S., Slot, J. W., Geuze, H. J. and Unanue,
E. R., 1991, Liposome-encapsulated antigens are processed in
lysosomes, recycled and presented to T-cells. Cell , 64, 393 /401.
Harris, E. and Cardelli, J., 2002, RabD, a dictyostelium Rab14related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion. Journal of Cell Science , 115, 3703 /
3713.
Hilden, S. A. and Madias, N. E., 1989, H /Ca2 exchange in rabbit
renal cortical endosomes. Journal of Membrane Biology , 112,
131 /138.
Hirst, J. and Robinson, M. S., 1998, Clathrin and adaptors.
Biochimica et Biophysics Acta , 1404, 173 /193.
Hirst, J., Futter, C. E. and Hopkins, C. R., 1998, The kinetics of
mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves
multivesicular endosomes without accumulating in a prelysosomal compartment. Molecular Biology of the Cell , 9, 809 /816.
Holroyd, C., Kistner, U., Annaert, W. and Jahn, R., 1999, Fusion of
endosomes involved in synaptic vesicle recycling. Molecular
Biology of the Cell , 10, 3035 /3044.
Holtzmann, E., 1989, Lysosomes (New York, London: Plenum
Press).
Honing, S., Griffith, J., Geuze, H. J. and Hunziker, W., 1996, The
tyrosine-based lysosomal targeting signal in lamp-1 mediates
sorting into Golgi-derived clathrin-coated vesicles. EMBO Journal , 15, 5230 /5239.
Huizing, M., Sarangarajan, R., Strovel, E., Zhao, Y., Gahl, Y. and
Boissy, R. E., 2001a, AP-3 mediates tyrosinase but not TRP-1
trafficking in human melanocytes. Molecular Biology of the Cell ,
12, 2075 /2085.
Huizing, M., Didier, A., Walenta, J., Anikster, Y., Gahl, W. A. and
Kramer, H., 2001b, Molecular cloning and characterization of
human VPS18, VPS 11, VPS16, and VPS33. Gene , 264, 241 /
247.
Hunziker, W. and Geuze, H. J., 1996, Intracellular trafficking of
lysosomal membrane proteins. Bioessays , 18, 379 /389.
Ihrke, G., Gray, S. R. and Luzio, J. P., 2000, Endolyn is a mucin-like
type I membrane protein targeted to lysosomes by its cytoplasmic
tail. Biochemical Journal , 345, 287 /296.
Jahraus, A., Egeberg, M., Hinner, B., Habermann, A., Sackman, E.,
Pralle, A., Faulstich, H., Rybin, V., Defacque, H. and Griffiths, G.,
2001, ATP-dependent membrane assembly of F-actin facilitates
membrane fusion. Molecular Biology of the Cell , 12, 155 /170.
Jahraus, A., Tjelle, T. E., Berg, T., Habermann, A., Storrie, B.,
Ullrich, O. and Griffiths, G., 1998, In vitro fusion of phagosomes
with different endocytic organelles from J774 macrophages.
Journal of Biological chemistry , 273, 30379 /30390.
Jones, E. W., 1977, Proteinase mutants of Saccharomyces cerevisiae . Genetics , 85, 23 /33.
Katzmann, D. J., Babst, M. and Emr, S. D., 2001, Ubiquitindependent sorting into the multivesicular body pathway requires
the function of a conserved endosomal protein sorting complex,
ESCRT-I. Cell , 106, 145 /155.
Kim, B. Y., Kramer, H., Yamamoto, A., Kominami, E., Kohsaka, S.
and Akazawa, C., 2001, Molecular characterization of mammalian
homologues of class C Vps proteins that interact with syntaxin-7.
Journal of Biological Chemistry , 276, 29393 /29402.
Kleijmeer, M., Ramm, G., Schuurhuis, D., Griffith, J., Rescigno, M.,
Ricciardi-Castagnoli, P., Rudensky, A. Y., Ossendorp, F., Melief,
C. J., Stoorvogel, W. and Geuze, H. J., 2001, Reorganization of
multivesicular bodies regulates MHC class II antigen presentation
by dendritic cells. Journal of Cell Biology , 155, 53 /63.
Klionsky, D. J., 1997, Protein transport from the cytoplasm to the
vacuole. Journal of Membrane Biology , 157, 105 /115.
Klionsky, D. J., Herman, P. K. and Emr, S. D., 1990, The fungal
vacuole: composition, function and biogenesis. Microbiology
Reviews , 54, 266 /292.
Ko, D. C., Gordon, M. D., Jin, J. Y. and Scott, M. P., 2001, Dynamic
movements of organelles containing Niemann-Pick Cl protein:
NPC1 involvement in late endocytic events. Molecular Biology of
the Cell , 12, 601 /614.
Kornfeld, S. and Mellman, I., 1989, The biogenesis of lysosomes.
Annual Review of Cell Biology , 5, 483 /525.
LaPlante, J. M., Falardeau, J., Sun, M., Kanazirska, M., Brown, E.
M., Slaugenhaupt, S. A. and Vassilev, P. M., 2002, Identification
and characterization of the single channel function of human
mucolipin-1 implicated in mucolipidosis type IV, a disorder
affecting the lysosomal pathway. FEBS Letters , 532, 183 /187.
Lawrence, B. P. and Brown, W. J., 1992, Autophagic vacuoles
rapidly fuse with pre-existing lysosomes in cultured hepatocytes.
Journal of Cell Science , 102, 515 /526.
Le Borgne, R., Alconada, A., Bauer, U. and Hoflack, B., 1998, The
mammalian AP-3 adaptor-like complex mediates the intracellular
transport of lysosomal membrane glycoproteins. Journal of
Biological Chemistry , 273, 29451 /29461.
Lemons, R. M. and Thoene, J. G., 1991, Mediated calcium transport
by isolated human fibroblast lysosomes. Journal of Biological
Chemistry , 266, 14378 /14382.
Liscum, L., 2000, Niemann-Pick type C mutations cause lipid traffic
jam. Traffic , 1, 218 /225.
Luzio, J. P., Rous, B. A., Bright, N. A., Pryor, P. R., Mullock, B. M.
and Piper, R. C., 2000, Lysosome /endosome fusion and lysosome biogenesis. Journal of Cell Science , 113, 1515 /1524.
Marsh, M., 2001, Endocytosis (Oxford University Press:, Oxford).
Martinez, I., Chakrabarti, S., Hellevik, T., Morehead, J., Fowler, K.
and Andrews, N. W., 2000, Synaptotagmin VII regulates Ca2 dependent exocytosis of lysosomes in fibroblasts. Journal of Cell
Biology , 148, 1141 /1149.
Martinez-Arca, S., Alberts, P. and Galli, T., 2000, Clostridial
neurotoxin-insensitive vesicular SNAREs in exocytosis and endocytosis. Biology of the Cell , 92, 449 /453.
Matteoni, R. and Kreis, T. E., 1987, Translocation and clustering of
endosomes and lysosomes depends on microtubules. Journal of
Cell Biology , 105, 1253 /1265.
McNeil, P. L., 2002, Repairing a torn cell surface: make way,
lysosomes to the rescue. Journal of Cell Science , 115, 873 /879.
McNeil, P. L. and Terasaki, M., 2001, Coping with the inevitable: how
cells repair a torn surface membrane. Nature Cell Biology , 3,
E124 /E129.
Mellman, I., 1996, Endocytosis and molecular sorting. Annual
Review of Cell Development Biology , 12, 575 /625.
Mellman, I. and Warren, G., 2000, The road taken: past and future
foundations of membrane traffic. Cell , 100, 99 /112.
Meyer, C., Eskelinen, E. L., Guruprasad, M. R., von Figura, K. and
Schu, P., 2001, Mu 1A deficiency induces a profound increase in
MPR300/IGF-II receptor internalization rate. Journal of Cell
Science , 114, 4469 /4476.
Meyer, C., Zizioli, D., Lausmann, S., Eskelinen, E.-L., Hamann, J.,
Saftig, P., von Figura, K. and Schu, P., 2000, m1A-adaptindeficient mice: lethality, loss of AP-1 binding and rerouting of
mannose 6-phosphate receptors. EMBO Journal , 19, 2193 /
2203.
Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya,
Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y. and Yoshimori, T., 2001,
Dissection of autophagosome formation using Apg5-deficient
mouse embryonic stem cells. Journal of Cell Biology , 152,
657 /667.
Mukherjee, S., Ghosh, R. N. and Maxfield, F. R., 1997, Endocytosis.
Physiological Reviews , 77, 759 /803.
Müller, O., Bayer, M. J., Peters, C., Andersen, J. S., Mann, M. and
Mayer, A., 2002, The Vtc proteins in vacuole fusion: coupling NSF
activity to V(0) trans-complex formation. EMBO Journal , 21, 259 /
269.
Lysosome biogenesis
Mullins, C. and Bonifacino, J. S., 2001, The molecular machinery for
lysosome biogenesis. Bioessays , 23, 333 /343.
Mullock, B. M., Bright, N. A., Fearon, C. W., Gray, S. R. and Luzio, J.
P., 1998, Fusion of lysosomes with late endosomes produces a
hybrid organelle of intermediate density and is NSF dependent.
Journal of Cell Biology , 140, 591 /601.
Mullock, B. M., Smith, C. W., Ihrke, G., Bright, N. A., Lindsay, M.,
Parkinson, E. J., Brooks, D. A., Parton, R. G., James, D. E.,
Luzio, J. P. and Piper, R. C., 2000, Syntaxin 7 is localized to late
endosome compartments, associates with Vamp 8, and is
required for late endosome /lysosome fusion. Molecular Biology
of the Cell , 11, 3137 /3153.
Murphy, R. F., 1991, Maturation models for endosome and lysosome
biogenesis. Trends in Cell Biology , 1, 77 /82.
Nakamura, N., Yamamoto, A., Wada, Y. and Futai, M., 2000,
Syntaxin 7 mediates endocytic trafficking to late endosomes.
Journal of Biological Chemistry , 275, 6523 /6529.
Nara, A., Mizushima, N., Yamamoto, A., Kabeya, Y., Ohsumi, Y. and
Yoshimori, T., 2002, SKD1 AAA ATPase-dependent endosomal
transport is involved in autolysosome formation. Cell Structure
and Function , 27, 29 /37.
Nichols, B. J. and Lippincott-Scwartz, J., 2001, Endocytosis without
clathrin coats. Trends in Cell Biology , 11, 406 /412.
Noda, T., Suzuki, K. and Ohsumi, Y., 2002, Yeast autophagosomes:
de novo formation of a membrane structure. Trends in Cell
Biology , 12, 231 /235.
Obermuller, S., Kiecke, C., von Figura, K. and Honing, S., 2002, The
tyrosine motifs of Lamp 1 and LAP determine their direct and
indirect targetting to lysosomes. Journal of Cell Science , 115,
185 /194.
Owen, D. J. and Evans, P. R., 1998, A structural explanation for the
recognition of tyrosine-based endocytotic signals. Science , 282,
1327 /1332.
Peden, A. A., Rudge, R. E., Lui, W. W. and Robinson, M. S., 2002,
Assembly and function of AP-3 complexes in cells expressing
mutant subunits. Journal of Cell Biology , 156, 327 /336.
Peters, C. and Mayer, A., 1998, Ca /calmodulin signals the
completion of docking and triggers a late step of vacuole fusion.
Nature , 396, 575 /579.
Peters, C., Andrews, P. D., Stark, M. J. R., Cesaro-Tadic, S., Glatz,
A., Podtelejnikov, A., Mann, M. and Mayer, A., 1999, Control of
the terminal step of intracellular membrane fusion by protein
phosphatase 1. Science , 285, 1084 /1087.
Peters, C., Bayer, M. J., Buhler, S., Andersen, J. S., Mann, M. and
Mayer, A., 2001, Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature , 409,
581 /588.
Peyron, P., Maridonneau-Parini, I. and Stegmann, T., 2001, Fusion
of human neutrophil phagosomes with lysosomes in vitro:
involvement of tyrosine kinases of the Src family and inhibition
by mycobacteria. Journal of Biological Chemistry , 276, 35512 /
35517.
Pfeffer, S. R., 1999, Transport-vesicle targeting: tethers before
SNAREs. Nature Cell Biology , 1, E17 /E22.
Pfeffer, S. R., 2001, Membrane transport: retromer to the rescue.
Current Biology , 11, R109 /R111.
Pillay, C. S., Elliott, E. and Dennison, C., 2002, Endolysosomal
proteolysis and its regulation. Biochemical Journal , 363, 417 /
429.
Piper, R. C. and Luzio, J. P., 2001, Late endosomes: sorting and
partitioning in multivesicular bodies. Traffic , 2, 612 /621.
Piper, R. C., Bryant, N. J. and Stevens, T. H., 1997, The membrane
protein alkaline phosphatase is delivered to the vacuole by a route
that is distinct from the VPS-dependent pathway. Journal of Cell
Biology , 138, 531 /545.
Porat, A. and Elazar, Z., 2000, Regulation of intra-Golgi membrane
transport by calcium. Journal of Biological Chemistry , 275,
29233 /29237.
Preston, R. A., Manolson, M. F., Becherer, K., Weidenhammer, E.,
Kirkpatrick, D., Wright, R. and Jones, E. W., 1991, Isolation and
characterization of PEP3 , a gene required for vacuolar biogenesis. Molecular Cell Biology , 11, 5801 /5812.
153
Pryor, P. R., Buss, F. and Luzio, J. P., 2000b, Calcium, calmodulin
and the endocytic pathway, ELSO Gazette 2. http://www.the-elsogazette.org/magazines/issues2/reviews/reviews1 pr.asp
Pryor, P. R., Mullock, B. M., Bright, N. A., Gray, S. R. and Luzio, J.
P., 2000, The role of intra-organellar Ca2 in late endosome /
lysosome heterotypic fusion and in the reformation of lysosomes
from hybrid organelles. Journal of Cell Biology , 149, 1053 /1062.
Puertollano, R., Aguilar, R. C., Gorshkova, I., Crouch, R. J. and
Bonifacino, J. S., 2001, Sorting of mannose 6-phosphate receptors mediated by the GGAs. Science , 292, 1712 /1716.
Raiborg, C., Bache, K. G., Gillooly, D. J., Madshus, I. H., Stang, E.
and Stenmark, H., 2002, Hrs sorts ubiquitinated proteins into
clathrin-coated microdomains of early endosomes. Nature Cell
Biology , 4, 394 /398.
Reaves, B. J., Banting, G. and Luzio, J. P., 1998, Lumenal and
transmembrane domains play a role in sorting type I membrane
proteins on endocytic pathways. Molecular Biology of the Cell , 9,
1107 /1122.
Reddy, A., Caler, E. V. and Andrews, N. W., 2001, Plasma
membrane repair is mediated by Ca2 -regulated exocytosis of
lysosomes. Cell , 106, 157 /169.
Reddy, J. V. and Seaman, M. N. J., 2001, Vps26p, a component of
retromer, directs the interactions of Vps35p in endosome-to-Golgi
retrieval. Molecular Biology of the Cell , 12, 3242 /3256.
Reggiori, F. and Pelham, H. R., 2001, Sorting of proteins into
multivesicular bodies: ubiquitin-dependent and -independent
targeting. EMBO Journal , 20, 5176 /5186.
Rieder, S. E. and Emr, S. D., 1997, A novel RING finger protein
complex essential for a late step in protein transport to the yeast
vacuole. Molecular Biology of the Cell , 8, 2307 /2327.
Robinson, J. S., Klionsky, D. J., Banta, L. M. and Emr, S. D., 1988,
Protein sorting in Saccharomyces cerevisiae : isolation of mutants
defective in the delivery and processing of multiple vacuolar
hydrolases. Molecular Cell Biology , 8, 4936 /4948.
Robinson, M. S. and Bonifacino, J. S., 2001, Adaptor-related
proteins. Current Opinions in Cell Biology , 13, 444 /453.
Rodriguez, A., Webster, P., Ortego, J. and Andrews, N. W., 1997,
Lysosomes behave Ca2 -regulated exocytic vesicles in fibroblasts and epithelial cells. Journal of Cell Biology , 137, 93 /104.
Rothman, J. E., 1994, Mechanisms of intracellular protein transport.
Nature , 372, 55 /63.
Rothman, J. H. and Stevens, T. H., 1986, Protein sorting in yeast:
mutants defective in vacuole biogenesis mislocalize vacuolar
proteins into the late secretory pathway. Cell , 47, 1041 /1051.
Rous, B. A., Reaves, B. T., Ihrke, G., Briggs, J. A. G., Gray, S. R.,
Stephens, D. J., Banting, G. and Luzio, J. P., 2002, The role of the
adaptor complex AP-3 in targeting wild type and mutated CD63 to
lysosomes. Molecular Biology of the Cell , 13, 1071 /1082.
Sachse, M., Urbe, S., Oorschot, V., Strous, G. J. and Klumperman,
J., 2002, Bilayered clathrin coats on endosomal vacuoles are
involved in protein sorting toward lysosomes. Molecular Biology of
the Cell , 13, 1313 /1328.
Sato, T. K., Rehling, P., Peterson, M. R. and Emr, S. D., 2000, Class
C Vps protein complex regulates vacuolar SNARE pairing and is
required for vesicle docking/fusion. Molecular Cell , 6, 661 /671.
Scheenen, W. J. J. M., Wollheim, C. B., Pozzan, T. and Fasolato, C.,
1998, Ca2 depletion from granules inhibits exocytosis. Journal
of Biological Chemistry , 273, 19002 /19008.
Seals, D. F., Eitzen, G., Margolis, N., Wickner, W. T. and Price, A.,
2000, A Ypt/Rab effector complex containing the Sec I homolog
Vps33p is required for homotypic vacuole fusion. Proceedings of
the National Academy of Sciences (USA) , 97, 9402 /9407.
Seaman, M. N. J. and Luzio, J. P., 2001, Lysosomes and other late
compartments of the endocytic pathway. In Endocytosis , M.
Marsh, ed. (Oxford University Press, Oxford), pp. 111 /148.
Seaman, M. N. J., McCaffery, J. M. and Emr, S. D., 1998, A
membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. Journal of Cell Biology , 142, 665 /681.
Seeley, E. S., Kato, M., Margolis, N., Wickner, W. and Eitzen, G.,
2002, Genomic analysis of homotypic vacuole fusion. Molecular
Biology of the Cell , 13, 782 /794.
154
J.P. Luzio et al.
Sevrioukov, E. A., He, J.-P., Moghrabi, N., Sunio, A. and Kramer, H.,
1999, A role for deep orange and carnation eye-color genes in
lysosomal delivery in Drosophila . Molecular Cell , 4, 479 /486.
Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J. M., Brech, A.,
Callaghan, J., Toh, B. H., Murphy, C., Zerial, M. and Stenmark,
H., 1998, EEA1 links PI(3)K function to Rab5 regulation of
endosome fusion. Nature , 394, 494 /498.
Simpson, F., Bright, N. A., West, M. A., Newman, L. S., Darnell, R. B.
and Robinson, M. S., 1996, A novel adaptor-related protein
complex. Journal of Cell Biology , 133, 749 /760.
Srivastava, A., Woolford, C. A. and Jones, E. W., 2000, Pep3p/
Pep5p complex: a putative docking factor at multiple steps of
vesicular transport to the vacuole of Saccharomyces cerevisiae .
Genetics , 156, 105 /122.
Stepp, J. D., Huang, K. and Lemmon, S. K., 1997, The yeast adaptor
protein complex, AP-3, is essential for the efficient delivery of
alkaline phosphatase by the alternate pathway to the vacuole.
Journal of Cell Biology , 39, 1761 /1774.
Stoorvogel, W., Kleijmeer, M. J., Geuze, H. J. and Raposo, G., 2002,
The biogenesis and functions of exosomes. Traffic , 3, 321 /330.
Stoorvogel, W., Strous, G. J., Geuze, H. J., Oorschot, V. and
Schwartz, A. L., 1991, Late endosomes derive from early
endosomes by maturation. Cell , 65, 417 /427.
Storrie, B. and Desjardins, M., 1996, The biogenesis of lysosomes: is
it a kiss and run continuous fusion and fission process?
BioEssays , 18, 895 /903.
Sullivan, K. M. C., Busa, W. B. and Wilson, K. L., 1993, Calcium
mobilization is required for nuclear vesicle fusion in vitro :
implications for membrane traffic and IP3 receptor function.
Cell , 73, 1411 /1422.
Sun, M., Goldin, E., Stahl, S., Falardeau, J. L., Kennedy, J. C.,
Acierno Jr., J. S., Bove, C., Kaneski, C. R., Nagle, J., Bromley, M.
C., Colman, M., Schiffmann, R. and Slaugenhaupt, S. A., 2000,
Mucolipidosis type IV is caused by mutations in a gene encoding
a novel transient receptor potential channel. Human Molecular
Genetics , 9, 2471 /2478.
Sutton, R. B., Fasshauer, D., Jahn, R. and Brunger, A. T., 1998,
Crystal structure of a SNARE complex involved in synaptic
exocytosis at 2.4 angstrom resolution. Nature , 395, 347 /353.
Thery, C., Zitvogel, L. and Amigorena, S., 2002, Exosomes:
composition, biogenesis and function. Nature Reviews in Immunology , 2, 569 /579.
Tooze, S. A., 1998, Biogenesis of secretory granule in the transGolgi network of neuroendocrine and endocrine cells. Biochimica
et Biophysica Acta , 1404, 231 /244.
Traub, L. M., Bannykh, S. I., Rodel, J. E., Aridor, M., Balch, W. E.
and Komfeld, S., 1996, AP-2-containing clathrin coats assemble
on mature lysosomes. Journal of Cell Biology , 135, 1801 /1814.
van Deurs, B., Holm, P. K., Kayser, L. and Sandvig, K., 1995,
Delivery to lysosomes in the human carcinoma cell line HEp-2
involves an actin filament-facilitated fusion between mature
endosomes and preexisting lysosomes. European Journal of
Cell Biology , 66, 309 /323.
Wade, N., Bryant, N. J., Connolly, L. M., Simpson, R. J., Luzio, J. P.,
Piper, R. C. and James, D. E., 2001, Syntaxin 7 complexes with
mouse Vps10p tail interactor 1b, syntaxin 6, vesicle-associated
membrane protein (VAMP)8, and VAMP7 in b16 melanoma cells.
Journal of Biological Chemistry , 276, 19820 /19827.
Wang, L., Seeley, E. S., Wickner, W. and Merz, A. J., 2002, Vacuole
fusion at a ring of vertex docking sites leaves membrane
fragments within the organelle. Cell , 108, 357 /369.
Ward, D. M., Leslie, J. D. and Kaplan, J., 1997, Homotypic lysosome
fusion in macrophages: analysis using an in vitro assay. Journal
of Cell Biology , 139, 665 /673.
Ward, D. M., Pevsner, J., Scullion, M. A., Vaughn, M. and Kaplan, J.,
2000, Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimidesensitive factor attachment protein receptors required for late
endosome /lysosome and homotypic lysosome fusion in alveolar
macrophages. Molecular Biology of the Cell , 11, 2327 /2333.
Wettey, F. R., Hawkins, S., Stewart, A., Luzio, J. P., Howard, J. C.
and Jackson, A. P., 2002, Controlled elimination of clathrin heavychain expression in DT40 lymphocytes. Science , 297, 1521 /
1525.
Wickner, W., 2002, Yeast vacuoles and membrane fusion pathways.
EMBO Journal , 21, 1241 /1247.
Woolley, K. and Martin, P., 2000, Conserved mechanisms of repair:
from damaged single cells to wounds in multicellular tissues.
Bioessays , 22, 911 /919.
Wurmser, A. E., Sato, T. K. and Emr, S. D., 2000, New component of
the vacuolar class C-Vps complex couples nucleotide exchange
on the Ypt7 GTPase to SNARE dependent docking and fusion.
Journal of Cell Biology , 151, 551 /562.
Yamamoto, A., Tagawa, Y., Yoshimori, T., Moriyama, Y., Masaki, R.
and Tashiro, Y., 1998, Bafilomycin A1 prevents maturation of
autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E. Cell
Structure and Function , 23, 33 /42.
Ybe, J. A., Brodsky, F. M., Hofmann, K., Lin, K., Liu, S. H., Chen, L.,
Earnest, T. N., Fletterick, R. J. and Hwang, P. K., 1999, Clathrin
self-assembly is mediated by a randomly repeated superhelix.
Nature , 399, 371 /375.
Zhu, Y., Doray, B., Poussu, A., Lehto, V. P. and Kornfeld, S., 2001,
Binding of GGA2 to the lysosomal enzyme sorting motif of the
mannose 6-phosphate receptor. Science , 292, 1716 /1718.
Received 3 September 2002; and in revised form
10 January 2003.