[Autophagy 4:1, -15; 1 January 2008]; ©2008 Landes Bioscience
Review
Yeast vacuole fusion
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A model system for eukaryotic endomembrane dynamics
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Department of Biology, Biochemistry Section; University of Osnabrück; Osnabrück, Germany
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(Vps, Pep), vacuole inheritance (Vac), and vacuole morphology
(Vam) in yeast (reviewed in ref. 1). In many identified mutants,
vacuole morphology is strongly perturbed. A morphology‑based
classification distinguishes normal (class A), partly (class B) and
highly (class C) fragmented vacuoles from large (class D) vacuoles,
and those having an enriched endosomal compartment (class E).2
The subsequent characterization of these genes unraveled the under‑
lying machinery of a number of trafficking pathways that are critical
for vacuole biogenesis, and in the case of the VAM genes, for vacuole
fusion.3 Below, these trafficking pathways are briefly described.
The vacuolar carboxypeptidase Y (CPY/Prc1) travels from the
trans‑Golgi‑Network (TGN) to the late endosome, from where it
is subsequently delivered to the vacuole.4,5 At the TGN, it is recog‑
nized by the receptor protein Vps10,6 concentrated and packed into
vesicles that pinch off from the TGN in an AP‑1/clathrin‑dependent
manner. After fusion with the late endosomal compartment, Prc1
dissociates from its receptor and is then transported to the vacuole
upon endosomal maturation. Vps10 is subsequently recycled back
to the TGN with the help of the retromer complex.7 Various other
hydrolases have additionally been shown to interact with Vps10 and
therefore follow the same route to the vacuole.8,9 Consequently, this
generic pathway has been termed the CPY‑pathway after its most
prominent cargo. However, a small subgroup of vacuolar resident
proteins, namely the PHO8 gene product alkaline phosphatase
(ALP), the SNAREs Vam3 and Nyv1 and the vacuolar casein kinase
Yck3, which is palmitoylated at its C terminus, travel to the vacuole
in a more direct trafficking event omitting the endosome.10‑13 This
pathway was termed the ALP‑ or AP‑3‑pathway after the adaptor
complex which is involved in the generation of vesicles at the TGN
in a clathrin‑independent fashion.11,14‑16 Additional pathways are
linked to the vacuole. The cytoplasm to vacuole targeting (Cvt)
pathway and macroautophagy use a similar set of components to
transport proteins to the vacuole.17‑21 Whereas the Cvt pathway
is a biosynthetic route to direct aminopeptidase I (Ape1) to the
vacuole lumen, macroautophagy is a catabolic pathway, which plays
a crucial role for cell survival upon starvation. In both cases, a double
membrane of unknown origin engulfs organelles and cytosolic mate‑
rial (macroautophagy) or oligomeric Ape1 (Cvt pathway) and targets
them directly to the yeast vacuole.17
Considering the number of trafficking pathways leading to the
vacuole, it is not surprising that the vacuole is a highly dynamic
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Vesicular transport in eukaryotic cells is concluded with the
consumption of the vesicle at the target membrane. This fusion
process relies on Rabs, tethers and SNAREs. Powerful in vitro
fusion systems using isolated organelles were crucial to obtain
insights into the underlying mechanism of membrane fusion—
from the initiation of fusion to lipid bilayer mixing. Among these
systems, yeast vacuoles turned out to be particularly useful as they
can be manipulated biochemically and genetically. Studies relying
on this organelle have revealed insights into the connection of
vacuole fusion to endomembrane biogenesis. A number of fusion
factors were identified and characterized over the last several years,
and placed into the fusion cascade. Within this review, we will
present and discuss the current state of our knowledge on vacuole
fusion.
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Key words: vacuole, lysosome, fusion, SNARE, HOPS, endomembrane
Introduction
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The yeast vacuole, which resembles the lysosome of higher
eukaroytes, is positioned at a crucial point of the eukaryotic endo‑
membrane system. The two major trafficking pathways in the cell,
the endocytic and the biosynthetic/secretory pathway target cargo to
the vacuole/lysosome (Fig. 1). The metabolic function of the vacuole/
lysosome has often been referred to as the cell’s “stomach” or “sink”.
Nowadays, and to shed a more realistic light on this organelle, one
would rather call it the major recycling facility of the cell. Its acidic
pH is a prerequisite for the activity of the numerous hydrolases, i.e.,
nucleases, phosphatases, lipases and proteases within the vacuolar
lumen, and the low molecular weight degradation products of these
enzymes’ catabolic activities are released into the cytosol for further
use. Moreover, lysosomes are critical during starvation and for the
degradation of cell surface receptors, thus allowing cells to respond
to extracellular signals and adjust their response.
Research on vacuole biogenesis and function was strongly promoted
by several elegant genetic screens, which led to the identification of
proteins involved in endocytosis (End), biosynthetic transport
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This manuscript has been published online, prior to printing.Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Clemens W. Ostrowicz, Christoph T.A. Meiringer and Christian Ungermann*
*Correspondence to: Christian Ungermann; University of Osnabrück Department
of Biology; Biochemistry Section; Barbarastrasse 13; Osnabrück 49076 Germany;
Tel.: +49.541.969.2752; Fax: +49.541.969.2884; Email: Christian.Ungermann@
biologie.uni-osnabrueck.de
Submitted: 07/07/07; Revised: 09/10/07; Accepted: 09/12/07
Previously published online as an Autophagy E-publication:
www.landesbioscience.com/journals/autophagy/article/5054
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Figure 1. Trafficking pathways leading to the yeast vacuole. For details see text. TGN, trans‑ Golgi network; EE, early endosome; LE, late endosome; MVB,
multivesicular body.
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organelle.22 During cell division the vacuole fragments partially,
producing vesicular and tubular structures, which are transported
into the emerging bud. In order to control the overall shape of the
vacuolar organelle, inherited vacuolar vesicles fuse later during cell
division, maintaining a low copy number for this organelle.23‑25 A
number of proteins involved in inheritance have been identified in
the past, including the vacuolar protein Vac8, the actin‑binding Myo2
protein and its adaptor, Vac17.22 Moreover, the dynamin‑homolog
Vps1 has been implicated in vacuole fission.26,183 Vacuole fragmen‑
tation and fusion is also connected to osmoregulation, and to the
synthesis and turnover of phosphatidylinositol‑(3,5)‑bisphosphate
(PI(3,5)P2).27‑30 Nevertheless, a clear concept of vacuole fission and
fragmentation is still missing.
Yeast vacuoles provide several important advantages to analyze
fusion in detail, including the excellent genetic and biochemical
tools available for S. cerevisiae, the facile purification of vacuoles,
fluorescent techniques to follow vacuoles in vivo and in vitro, and
an established in vitro fusion assay.31‑33 Within this review, we
will discuss the present knowledge on vacuole fusion, and on the
SNAREs, tethers and additional proteins involved in the fusion
process. Furthermore, we will integrate findings on vacuole fusion
with the knowledge on fission, trafficking pathways to the vacuole,
and vacuole biogenesis.
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The in vitro vacuole fusion assay. Analysis of vacuole fusion was
greatly facilitated by the invention of an in vitro fusion assay.25,34
For this assay, vacuoles from two yeast strains with a different genetic
background are employed. One of these strains lacks the major phos‑
phatase Pho8, which usually serves as a degradative enzyme cleaving
phosphoester bonds in the lumen of the vacuole. The other strain
lacks the major vacuolar protease Pep4, which is needed for the acti‑
vation of other hydrolases, including Pho8; it therefore accumulates
inactive pro‑Pho8. Upon mixing of the vacuoles purified from the
two strains in a fusion reaction, the vacuoles fuse, and Pep4 removes
the propetide from Pho8. Fusion activity can then be measured
using p‑nitrophenylphosphate as a Pho8 substrate, which becomes
yellow upon dephosphorylation. Thus, the amount of generated
p‑nitrophenol can be directly linked to the rate of fusion between
the two pools of vacuoles.
The advantages of this in vitro fusion assay are manifold: It is
relatively easy to perform and basically every strain with the depicted
background can be assayed for fusion activity, allowing for the
efficient screening of fusion relevant ORFs. Furthermore, chemical
compounds and proteins such as antibodies and fusion factors can
simply be added to the reaction to investigate their influence on the
fusion rate. However, since the assay employs biological reaction
partners certain shortcomings are unavoidable: Mutants with altered
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Vacuole fusion
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Figure 2. Stages of in vitro vacuole fusion. The main reactions involving Ypt7, HOPS and SNAREs are indicated below the cartoon. Factors implicated in
subreactions and regulation processes are listed in a second level. PIP, PI(3)P; PIP2, PI(3,5)P2; DAG, diacylglycerol.
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vacuole morphology may have vacuoles deficient in Pho8 or Pep4,
since both marker proteins have to be sorted to the vacuole, and
some endosomal proteins affect vacuole morphology.2,35 Inhibitors
applied in the assay may inhibit Pho8 or Pep4 activity rather than the
targeted fusion factor.36,37 Usually, the latter is controlled by lysing
vacuoles prior to fusion in the presence of the inhibitor, such that
Pep4‑dependent activation of Pho8 can be measured independent
of fusion.
An alternative content mixing assay has been presented recently.
For this assay, two segments of b‑lactamase were fused to the tran‑
scription factors Fos or Jun and targeted to the vacuole, where
the formation of the Fos‑Jun complex reconstitutes b‑lactamase
activity.37 This assay shows the same kinetic properties as the
Pho8‑based assay, but b‑lactamase activity seems to be less sensitive
to inhibitors directed against fusion factors. In addition, lipid mixing
assays have been developed, which allow a resolution of lipid and
content mixing.37,38
In some studies, the in vitro fusion assays have been complemented
by the visual examination of vacuole fusion under the fluorescence
microscope.36,37,39‑41 Fused vacuoles increase in volume and popula‑
tions can then be analyzed. In vivo vacuole fusion during osmotic
stress is used as an additional measure. If exposed to hyperosmotic
stress, vacuoles fragment, but fuse again, if the cells are subsequently
placed into low osmotic medium afterwards.29,42 Neither alternative
assay can be easily manipulated nor permit a kinetic analysis of the
fusion reaction, but they may be valuable to complement the content
and lipid mixing assay. In the following sections, we will discuss the
present knowledge on vacuole fusion factors and give insight into
the fusion mechanism and its regulation.
Stages of Vacuole Fusion
A series of events occurs prior to lipid bilayer mixing (Fig. 2).
It is still under debate if distinct steps follow a defined order or if
the fusion rather arises through cooperative action of fusion factors
involved in different steps of the reaction.43 It seems plausible that
fusion occurs at least partly in a consecutive order of events, since
inhibitors to intermediate steps will block the subsequent fusion
reaction.34,44 As observed in the in vitro vacuole fusion assay, these
steps are termed priming, tethering, docking and fusion/bilayer
mixing32 and will be introduced below.
Priming. In the ATP‑dependent priming reaction, vacuoles are
prepared for the first contact and subsequent fusion. A number of
fusion factors that reside on the vacuole in an inactive state are now
activated in an ATP‑dependent manner.39,45‑48 The NSF‑homolog
Sec18, an AAA‑ATPase, disassembles cis‑SNARE complexes with the
help of its co-factor Sec17, which is released from vacuoles during
the priming reaction.46 In this reaction, the soluble SNARE Vam7
dissociates from the complex but stays partially bound to the vacuole
membrane.49,50 In addition, the dynamin‑like Vps1 protein, which
binds to Vam3 on vacuoles, is released from this site.26 After disas‑
sembly, SNAREs are in an active, fusion‑competent state. A couple
of additional reactions such as palmitoylation of the Vac8 fusion
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SNAREs. SNAREs are membrane‑anchored proteins that share
a conserved coiled‑coil domain adjacent to their transmembrane
domain, which is required for regulated fusion of lipid bilayers
(reviewed in refs. 56, 67, 75 and 76). They are classified as either
Q‑ or R‑SNAREs, depending on whether they contain a glutamine
or arginine residue at a conserved position within their SNARE
domain.77,78 According to their localization, they are also referred
to as vesicle (v)‑ and target membrane (t) ‑SNAREs.79 Most
R‑SNAREs are acting as v‑SNAREs, while most Q‑SNAREs form
t‑SNARE complexes. The fusion‑competent assembly of SNAREs
from opposing membranes, termed the trans‑SNARE complex or
SNAREpin,64 in general consists of three Q‑ and one R‑SNARE.
Once the bilayers have fused and all SNAREs are located within
the same membrane, the still‑assembled SNARE-complex is termed
a cis‑SNARE complex. SNAREs are recycled for further rounds of
fusion by disassembly of the cis‑SNARE complex by NSF/Sec18 and
a‑SNAP/Sec17.80,81
Studies on vacuole fusion clarified the order of events leading
to membrane fusion, when it became clear that the yeast NSF
and a‑SNAP‑homologs Sec18 and Sec17 act prior to docking
and not during fusion.39,46 Later on, five SNAREs were identified
on yeast vacuoles that are involved in the fusion reaction: three
Q‑SNAREs (Vam3, Vam7 and Vti1) and two R‑SNAREs (Ykt6
and Nyv1).47,49,82‑85 The Q‑SNAREs Vam3, Vam7 and Vti1 form
complexes either with Nyv1 or Ykt6.57,60 Whereas the Nyv1 complex
is driving homotypic vacuole fusion, the Ykt6 complex is implicated
in other fusion reactions at the vacuole86 and palmitoylation.87
Priming. Every vacuole fusion event leads to the accumulation
of the SNARE-complex on the same membrane‑the cis‑SNARE
complex, which is associated with Sec1747,88 and the HOPS
complex.48,89 For further rounds of fusion to occur, the cis‑SNARE
complex is disassembled by the ATPase Sec18,47 which leads to the
release of Sec17,46,88 and the HOPS complex48 as well as the soluble
SNARE Vam7.49,50,90 Vam7 lacks a transmembrane domain, but
contains a phosphoinositide‑3‑phosphate (PI(3)P) binding module,
a PX domain, at its N terminus, which is required for membrane
binding.91‑93 Following its release, Vam7 is specifically recruited to
the docking site.50 Moreover, Vam7 initiates docking of vacuoles in
conjunction with Ypt7,90 while the free Q‑SNARE Vam3 engages
in binding to the HOPS complex,89,94 which subsequently leads to
the docking of vacuoles. It is possible that the N‑terminal domain
of Vam3 has a critical role in this process. Mutant vacuoles carrying
Vam3 without this domain show reduced trans‑SNARE pairing,
HOPS binding and fusion.89 It should be noted, however, that
another study did not find impaired fusion in a similar mutant.95
SNAREs, tethering, docking and fusion. The events taking
place during vacuole fusion downstream of priming can be divided
into two steps: (1) the reversible tethering of vacuoles mediated by
HOPS and (2) the irreversible pairing of SNAREs between vacuoles
in trans. Initial tethering of vacuoles occurs at specific sites and is
mediated by the Rab Ypt7, but apparently occurs independent of
SNARE function.40,53,70 Following this, Vam3 and Vam7 regulate
each other’s enrichment at these initial contact sites, whereas Vti1
assembly seems to be regulated by a different mechanism.70 Ypt7 is
thought to orchestrate the associations between the Q‑SNARE and
HOPS thereby bridging the transient tethering with the irreversible
step of trans‑SNARE complex formation.96 Lipid rearrangements are
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factor and Vam10 function (see below) have also been connected to
priming.51,52
Tethering. Tethering—the reversible but distinct contact between
vacuoles—is dependent on the fully assembled HOPS tethering
complex and the small Rab‑GTPase Ypt7, present in its GTP‑form
on both membranes.39,53 The HOPS complex is a large hexameric
protein complex, consisting of the four C‑Vps proteins (Vps11, 16,
18, 33), Vps41 and Vam6.54,55 The complex has a molecular mass
of approximately 700 kDa, and may be present in an even larger
complex on membranes.48 Compared to the much smaller SNARE
complex, the large size of the HOPS complex is believed to enable it
to reach over much bigger distances, which predestines it for medi‑
ating first contact between vacuoles.56 At present, it is not clear if
the bridging by one HOPS complex is sufficient for efficient vacuole
tethering or whether the HOPS complex dimerizes during tethering.
A major problem in addressing the tethering stage in the vacuole
fusion assay is to dissect tethered from docked vacuoles, which
already contain trans‑SNARE complexes.
Docking and fusion. During docking, unpaired SNAREs that
were made available during vacuole priming assemble into trans‑com‑
plexes between opposing membranes. According to several studies, it
seems that the Q‑SNAREs Vam3, Vam7 and Vti1 on one membrane
pair with the R‑SNARE Nyv1 from the other membrane.53,57‑59
This arrangement is sufficient to drive liposome fusion using purified
SNAREs.60 The proteins form a zipper‑like complex, which propa‑
gates from the N‑terminal, cytoplasmic side to the transmembrane
proximal part of the SNARE domain.61‑63 Most likely, assembly of
the complex creates sufficient physical constraints on the vacuolar
membrane to drive lipid bilayer mixing.64‑67 It should be noted that
the liposome fusion assay, which has been employed to demonstrate
SNARE-dependent fusion in vitro, is dependent on the vesicle prepa‑
ration and SNARE density on the liposome.68,69 It therefore seems
likely that additional factors promote SNARE-mediated fusion
in vivo. Furthermore, inhibitors and mutants that allow SNARE
pairing, but block lipid mixing have been described in the litera‑
ture.37,38,59,101 It is presently unclear how vacuole fusion is triggered
once the fusion process is initiated; the evidence obtained on addi‑
tional factors implicated in this reaction will be discussed below.
A number of factors such as the HOPS complex, the SNAREs,
and Ypt7 specifically enrich on a structure surrounding the future
fusion site.40,70 The formation of the so‑called vertex‑ring leads to
a flattening of the vacuole boundaries and therefore enlarges the
contact zone between the fusion‑destined vacuoles. Fusion of vacu‑
oles was shown to leave membrane fragments within the organelle,
which leads to the formation of intralumenal membranes.40 The
concerted enrichment of several factors at the fusion site may support
the SNARE-driven fusion process. In vivo, such enrichment has, for
example, been shown for the HOPS subunit Vps41, the docking
factor Ccz1 and for the Vac8 fusion factor.71,72‑74
The Vacuole Fusion Machinery
Vacuole fusion depends on a number of conserved fusion factors.
We consider SNAREs, the Rab GTPase Ypt7, and the HOPS teth‑
ering factor as the basic machinery driving the fusion reaction, and
will therefore discuss them separately. Afterwards, we will focus on
additional proteins connected to the fusion reaction.
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impaired by the insertion of 12 amino acids, fusion was reduced to
background levels.95 Comparable to SNARE-mediated fusion at
other compartments within eukaryotes,67 the vacuolar trans‑SNARE
complex bridging two vacuoles must contain three Q‑(Vam3, Vti1,
Vam7) and one R‑SNARE (Nyv1) in the 0‑layer.58 It also depends
on specific lipids, which form distinct domains where SNAREs are
assembled.58
The R‑SNARE Ykt6. Ykt6 is an R‑SNARE involved in vacuole
fusion that was initially identified as being involved in Golgi trans‑
port steps107 and transport from the ER to the Golgi.108,109 Ykt6 is
the most highly conserved SNARE from yeast to humans and was
subsequently identified as a component of the vacuolar SNAREcomplex.84,86 Unlike most other SNAREs, with the exception of
Vam7 at the vacuole, Ykt6 is not anchored to the membrane via
a transmembrane domain, but shuttles between the cytosol and
membranes. Its C‑terminal CAAX‑box is prenylated at the distal
cysteine residue, whereas the adjacent cysteine is reversibly palmi‑
toylated. On membranes, Ykt6 is both prenylated and palmitoylated,
whereas it is believed to be depalmitoylated upon dissociation from
membranes.110‑112 In its cytosolic state, the N‑terminal longin‑do‑
main of Ykt6 is thought to fold back onto its C‑terminal coiled‑coil
domain, thereby enveloping its hydrophobic prenyl anchor.113 Ykt6
may regulate its own palmitoylation and depalmitoylation114 and
thereby determine its localization.110,111
The function of Ykt6 at the vacuole remains puzzling. It is
implicated to function in several trafficking pathways towards the
vacuole, i.e. the CPY pathway downstream of the endosome, as
well as the AP‑3 and Cvt pathways.86,115 But whether it functions
as a bona fide SNARE protein and a regulator is still unclear. To
complicate matters, Ykt6 can also functionally substitute for the
R‑SNARE Sec22 at the ER116 and is part of the SNARE complex
consisting of Pep12, Vti1 and Syn8 at the late endosome.86 At the
vacuole, Ykt6 forms a complex with the Q‑SNAREs Vam3, Vam7
and Vti1,57 and Ykt6 and Nyv1 can compete for this complex in
vitro.60 Furthermore, in vitro reconstitution assays have demon‑
strated that Ykt6 cannot function as a v‑SNARE, unless its prenyl
anchor is replaced by a membrane‑spanning peptide117 and that it
most likely is part of the Q‑SNARE complex sitting on the acceptor
membrane.118 Whether this is also the case for vacuole fusion, has
not yet been determined. Interestingly, adding high concentrations
of recombinant Vam7 to in vitro vacuole fusion reactions enables
Ykt6 to functionally substitute for Nyv1.119 This may indicate that
on isolated liposomes, SNAREs need to be stably anchored to the
membrane via a transmembrane domain, whereas in vivo, lipidic
anchors are sufficient to drive SNARE-mediated fusion of bilayers
in conjunction with their accessory proteins. Alternatively, Ykt6 may
not act as a v‑SNARE in fusion, but as part of a t‑SNARE complex.
Hence, the transmembrane proteins of the complex, Vti1 or Vam3,
may act as v‑SNAREs, whereas the other may form the t‑SNARE
complex with Vam7 and Ykt6. Ykt6 depalmitoylation and release
from vacuoles has been linked to Vac8 palmitoylation87,112 and will
be discussed below.
The dynamics of the Q‑SNARE Vam7. In a genetic screen for
vacuolar morphology, Vam7 was identified as a candidate protein
that when mutated exhibited missorting of cargo destined for the
vacuole.49,85,120 Vam7 is a SNAP‑25 homolog77 that binds to PI(3)P
on membranes via its PX domain (see above). Vam7 shows a genetic
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important for these events to proceed.40,70,93,97‑99 SNARE pairing
between vacuoles can be detected using SNARE deletion strains
or assays with tagged SNAREs.53,57,59 Each of these assays shows a
Sec17‑Sec18‑, ATP‑ and Ypt7‑dependent accumulation of SNARE
complexes, which are also detectable when fusion inhibitors are
added to the reaction.
Trans‑SNARE assays have been very helpful to define stages of
the fusion reaction, but also have certain shortcomings. Each assay
may detect both trans‑SNARE complexes, as well as newly formed
cis‑complexes, because inhibitors may be bypassed. Moreover, the
detection of trans‑SNARE complexes is performed by immunopre‑
cipitation after detergent lysis, which can promote complete SNARE
zippering subsequent to lysis. In the assay using vacuoles from
deletion strains, fusion can be blocked by previously characterized
inhibitors.53 Without inhibition, lower fusion rates are observed
compared to wild‑type vacuoles, whereas trans‑SNARE pairing can
be clearly detected in a Sec17‑Sec18‑, ATP‑ and Ypt7‑dependent
manner.53,57 Furthermore, trans‑SNARE pairs are also detected in
the presence of fusion inhibitors,100 and between vph1D vacuoles,
which do not fuse in vitro.101 Addition of Sec17‑Sec18 and ATP
to an ongoing fusion reaction at a time, when fusion was not yet
significant, but trans‑SNARE complexes were observed, reduces the
steady‑state amount of SNARE complexes below detection, which
suggests that SNARE complexes may be dispensable for the final
fusion step.53 It is, however, possible that the remaining trans‑SNARE
pairs, which escaped detection, drive the fusion reaction. In liposome
fusion with synaptic SNAREs, the Sec18‑Sec17 homologs NSF and
a‑SNAP show disassembly activity in the presence of ATP only when
cis‑SNARE complexes are present, suggesting that trans‑SNAREs per
se are insensitive to these chaperones unless fusion is completed.102
To date, it is unclear why the detected trans‑SNARE pairs in the
previously mentioned assay can be dissociated by Sec18‑Sec17
and ATP. Of note, the trans‑SNAREs formed between vacuoles
that contain tagged SNAREs57 are not sensitive to these reagents
(LaGrassa T, Rohde J, Ungermann C, unpublished observations). In
this second assay, trans‑SNAREs between vacuoles carrying tagged
SNAREs are formed as effectively as with the assay based on dele‑
tion strains, and fusion is similar to wild‑type.57 However, it cannot
be excluded that fusion is driven by the untagged SNAREs, which
are present as well. Recently, a third assay was devised to avoid this
problem. Here, nyv1D vacuoles were fused with vacuoles carrying
tagged Nyv1.59 In this case, trans‑SNAREs form in the previously
described manner and are detectable in the presence of a fusion
inhibitor MED (MARCKS effector domain), which binds the regu‑
latory lipid PI(4,5)P2.58,59 Although this assay provides an important
step forward, it will be necessary to develop additional approaches
that do not depend on any SNARE deletion strain and are capable
of dissecting sub‑steps in the SNARE assembly.
The release of Ca2+ from the vacuole lumen has been used as
an alternative assay to measure the requirement of trans‑SNARE
pairs for fusion.41 Indeed, fusion‑dependent release of Ca2+ also
occurs if vacuoles lacking Nyv1 are incubated with vam3D vacuoles,
which had previously been used for trans‑SNARE complex detec‑
tion.41,53,71,100,103,104 Fusion requires transmembrane‑anchored
Vam3,105 and fusion efficiency has been connected to the struc‑
ture of the Vam3 transmembrane domain.106 Furthermore, if the
coupling between transmembrane domain and SNARE domain was
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Indeed, binding of HOPS to the membrane is reduced in the absence
of Ypt7,48 but it has not been tested whether the identified affinity
of HOPS for phospholipids122 accounts for the residual association
of HOPS with vacuoles. Potentially, it is a combination of several
interactions, including its binding to SNAREs.89,94,122,138 HOPS
is thought to act as a tether, which spans the distance between two
vacuoles. This first contact between vacuoles seems to be reversible.53
Because Ypt7 appears to be required on both vacuoles,39 and HOPS
binds to Ypt7‑GTP,55 tethering could occur by transient dimerization
of HOPS. It is also possible that HOPS is binding asymmetrically to
SNAREs and Ypt7, and the symmetric requirement of Ypt7 is not
exclusively related to HOPS function. It is, however, likely that the
interaction of HOPS and SNAREs is crucial in the fusion pathway,
and potentially even for tethering. This point has been addressed
on several levels. First, HOPS binds to SNAREs, as outlined above.
Second, this binding is selective: SNAREs on vacuoles seem to bind
either HOPS or Sec17.96 Wickner and colleagues postulate that the
formed HOPS:SNARE complex is an intermediate towards fusion.
This likely explanation is, however, difficult to prove, as complexes
may not only occur on a pathway toward fusion, but also in a side
reaction. Recent results on vacuole and liposome fusion using puri‑
fied HOPS59 indicate that HOPS can proofread the SNARE 0‑layer
and is required to drive liposome fusion in the presence of SNAREs
(Wickner W, personal communication). Investigating the vertex ring
of docked vacuoles, it was also shown that HOPS, SNAREs and
Ypt7 accumulate in an interdependent manner at the docking site,70
indicating a crosstalk among, and potentially multimerization of,
these proteins. It should be noted that the enrichment of proteins at
the vertex site is challenging to quantify, and is only 1.5 to 2‑fold in
some cases.
Regulation of the HOPS complex function occurs on several
levels. During the fusion reaction, HOPS is released from vacu‑
oles,48 which might explain the ATP‑dependent dynamics of Vps33
observed in vivo and in vitro.134 Moreover, the membrane associa‑
tion of the HOPS complex is regulated by the vacuolar casein kinase
Yck3.71 Yck3 phosphorylates the HOPS subunit Vps41 on the vacu‑
olar membrane, which attenuates fusion in wild‑type conditions.
Yck3 knockout leads to a strong accumulation of Vps41 at vacuole
contact sites, whereas overexpression of the kinase strongly reduces
fusion. Recently, we identified a twin complex at the endosome,
called the CORVET complex, which shares the four class C subunits
with the HOPS complex.35 Overproduction of the Vps3 subunit of
the CORVET complex leads to a HOPS hybrid complex, in which
Vps3 replaces Vam6. It is therefore likely that HOPS assembly and
function is linked to endosome‑vacuole dynamics.
Additional proteins involved in fusion. Several proteins have
been implicated as important additional fusion factors, including
the vacuolar Vo/V1‑ATPase, Bem1, calmodulin, protein phosphatase
1, Vam10 and others. In most cases, fusion reactions are strongly
affected in a deletion mutant or in antibody inhibition experiments,
suggesting an intimate connection to vacuole fusion. Below, we will
discuss the proteins (in alphabetical order), the data obtained on the
function of these factors, and their potential role in vacuole fusion.
Bem1 and Cln3. During inheritance, vacuoles fuse as part of the
cell cycle. Therefore, it did not come as a surprise when the cyclin
Cln3 was implicated in vacuole fusion.139 Cln3 overproduction
results in vacuole fragmentation and vacuoles lacking Cln3 fuse
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interaction with Vam3 and physically interacts with Vam3 and
Sec18. In addition, a yeast two‑hybrid analysis suggests an interac‑
tion between Vam7 and the vacuolar Rab GTPase Ypt7.121
Vam7 is responsible for vacuolar SNARE complex integrity and
vacuole docking and is released from membranes in a Sec17‑Sec18‑
and ATP‑dependent manner.50,90 However, even after its release,
Vam7 is still required in vacuole fusion for Ypt7‑dependent docking90
and was shown to be specifically recruited back to the vacuole after
its release from the cis‑SNARE complex.50 Interestingly, cells lacking
Ypt7 have fragmented vacuoles that cannot fuse in vitro and have
little Vam7 on their vacuoles.90 The finding that recombinant
Vam7 added to in vitro fusion reactions can bypass the need for
Sec18‑Sec17‑mediated disassembly of the cis‑SNARE complex,
further supports the idea that Vam7 release and redocking are regula‑
tory and essential steps in vacuole fusion.119 Moreover, it indicates
that a sufficient amount of free SNAREs exists to allow for efficient
fusion. In addition to being responsible for SNARE integrity, Vam7
also binds to the HOPS complex122 suggesting that Vam7 may
couple activation of Ypt7 by HOPS during vacuole tethering to
SNARE complex assembly and subsequent fusion.
The HOPS tethering factor. Tethering during homotypic vacuole
fusion in yeast is mediated by the HOPS protein complex, which
consists of six subunits ranging from 79 to 123 kDa in size. The
complex is thought to mediate the first contact between vacuoles
by interacting with the Rab GTPase Ypt7.123 Four of its subunits
belong to the class C gene family, which result in the most frag‑
mented vacuole phenotype if deleted,2,124,125 whereas the two
additional subunits,Vps41/Vam2 and Vps39/Vam6, are class B
gene products.126 The HOPS complex can be purified as a 700
kDa hetero‑oligomeric complex that binds Ypt7 in its GTP‑bound
form.54,55,123
All six HOPS proteins have been characterized in some detail,
even though their precise function still needs further clarification.
Vps11/Pep5 and Vps18/Pep3 both contain essential C‑terminal
RING‑H2 domains. Mutations in the RING‑finger of Vps18
lead to vacuole fragmentation and missorting of proteins to the
vacuole,127 indicating an essential role of these domains in assembly
of the complex assembly or protein function. RING domains are
present in ubiquitin ligase proteins, and the mammalian Vps18
protein indeed promotes ubiquitination.128 Vps33 belongs to the
Sec1/Munc18 family, whose members bind SNAREs,129‑132 and
contains an ATP‑binding domain.133,134 Vps16 remains the least
characterized HOPS subunit, lacking a distinct domain topology.
The class B protein Vps41 has several N‑terminal WD‑40 domains,
which most likely form a b‑propeller structure.135 In addition to
its role in vacuole fusion, the protein is required for the formation
of AP‑3 vesicles at the late Golgi. In this context, Vps41 has been
discussed as a coat protein because specific mutants abolish AP‑3
vesicle generation.15,136 Vam6 has guanyl‑nucleotide exchange factor
(GEF) activity for Ypt7.54 Interestingly, all HOPS subunits (with
the exception of Vps33) have a common domain structure with an
N‑terminal b‑sheet and a long a‑helical C‑terminus (Ungermann
C, unpublished), consistent with the idea of a common ancestor or
convergent evolution.137
The mechanism of HOPS function on vacuoles is not resolved.
Based on the GEF activity of Vam6, it is believed that HOPS
activates Ypt7 at the vacuole and binds to it in the GTP‑bound form.
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studies. Multiple lines of evidence connect it to the fusion cascade.103
In fusion it may, however, have a role distinct from a Ca2+‑sensor, a
phenomenon that has been observed also in microautophagy, which
depends on a Ca2+‑independent function of calmodulin.156
Cdc42, Rho1 and the actin cytoskeleton. Several lines of evidence
link the cytoskeleton to vacuole fusion. Early work implicated micro‑
tubules in vacuole fusion,157 a connection that was not followed up
further. A more recent study surveyed yeast vacuole morphology
in all viable yeast deletion strains and identified several mutants
involved in actin dynamics and turnover.158 Follow‑up studies
confirmed that certain actin mutants, as well as antibodies directed
against components of the actin‑branching Arp2/3 complex such as
Las17 or Arp3 inhibit vacuole fusion.159 For fusion, actin‑depoly‑
merization is required prior to docking, whereas actin polymerization
seems to support fusion.159 It is likely that fusion‑dependent actin
dynamics on vacuoles are linked to two Rho‑GTPases, Cdc42 and
Rho1, that were implicated in independent studies.160,161 Mutants
in cdc42 and rho1 are defective in fusion and have perturbed vacuole
morphology, and antibodies to Cdc42 inhibit vacuole fusion in a
dose‑dependent manner.160,161 Rho‑GTPases can be extracted by
Rdi1, which inhibits vacuole fusion if GST‑tagged,160 but not if
purified without a tag.43 This observation is quite puzzling, since
both proteins seem to be equally active in removing Rho1 and Cdc42
from membranes,43 suggesting that GST‑Rdi1 does not inhibit the
Rho‑GTPases. Potentially, only a fraction of both proteins is required
to modulate the actin cytoskeleton. The mutant and antibody inhibi‑
tion results presented on Cdc42, Rho1, and actin suggest an active
role in fusion.
How then do Cdc42, Rho1, and actin act on vacuoles? Studies
in other fusion systems suggest an active involvement of actin (for
example, see refs. 162 and 163), and it will be interesting to learn,
whether actin provides a general membrane matrix or is actively
involved in specific sub‑reactions. Mapping interaction partners
during the fusion reaction should provide some insight into this
question. It should be mentioned that Cdc42 has also been encoun‑
tered in a different context; Cdc42 overexpression rescues the
fragmentation phenotype observed in cln3D cells,140 suggesting a
connection between cell cycle control and actin dynamics. Furthermore,
S. pombe Cdc42 is connected genetically to Vtc1/Nrf1,164 a factor
implicated in the fusion process (see below).
Enolase. Vacuole fusion is stimulated by cytosol or a cytosolic
extract. One of the stimulating activities purified from yeast cytosol
turns out to be enolase, a glycolytic enzyme involved in the genera‑
tion of phosphoenolpyruvate.165 Localization of enolase to vacuoles
requires the Band3‑homolog Bor1, and bor1D vacuoles are frag‑
mented and lack bona fide vacuolar fusion factors like Vam3 and
Ypt7. This phenotype is actually quite reminiscent of the depletion
of the acyl CoA binding protein (Acb1), which does not seem to be
directly connected to the fusion reaction.166 Generally, it appears
that vacuole dynamics are linked to the metabolic state of the cell
and potentially connected to autophagy.17 How sensing of nutrients
occurs at the vacuole is not yet known. Potentially, ubiquitination
of selected fusion factors is connected to this sensing mechanism.167
LMA1. LMA1 consists of two subunits, the inhibitor of proteinase
B (Pbi1 or IB2) and thioredoxin, and was initially purified from
cytosolic extracts that stimulate vacuole fusion.168,169 If added
to vacuoles, LMA1 binds to Vam3 on vacuoles (presumably via
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inefficiently. Subsequently, a protein required for cell polarization
during budding and mating, Bem1, was identified as a potential
target of Cln3‑mediated phosphorylation.140 Vacuoles lacking Bem1
are fragmented and deficient in fusion. Whereas one report identi‑
fies S72 as a potential phosphorylation site in Bem1,140 another
could not confirm that this residue is critical for Bem1 func‑
tion.141 Until now, the molecular function of Bem1 or additional
Cln3‑cyclin‑dependent kinase targets have not been clarified.
Ca2+/Calmodulin. During the arrival of an action potential at
the synapse, Ca2+‑channels open and allow Ca2+ to flush from the
extracellular medium into the presynapse, thus triggering exocy‑
tosis. The central sensor is synaptotagmin, a membrane protein
on synaptic vesicles, which binds Ca2+ via its two C2 domains.142
Another Ca2+‑binding protein, calmodulin, has also been discussed
in this context.143 Calmodulin is conserved and also found on yeast
vacuoles.144 It was implicated as a Ca2+ sensor as it can accumulate
on vacuoles and stimulates fusion if added to isolated vacuoles.100,144
In addition, certain mutant alleles of calmodulin have fragmented
vacuoles, and antibodies and calmodulin‑specific agents block
vacuole fusion.144 The connection of Ca2+ to the fusion event was
implied by the detection of a Ca2+‑flux that was released from vacu‑
oles during fusion, and fusion is indeed sensitive to the well‑known
Ca2+‑chelator BAPTA, but not to EGTA, which sequesters Ca2+ less
efficiently.41,144
In yeast, the vacuole represents a main Ca2+ reservoir, and two
transporters, Vcx1 and Pmc1, pump Ca2+ into the vacuole.145
The Pmc1 Ca2+‑ATPase binds to, and is inhibited by, the SNARE
Nyv1,146 though fusion and Ca2+‑flux occurs in the absence of Vcx1
and Pmc1.41,100 It should be noted that fusion events among tester
vacuoles carrying the same reporter will be detected as a Ca2+‑flux,
but cannot be detected by a content mixing assay.36,41 During the
progression of fusion, this transient flux vanishes quickly,144 prob‑
ably due to the rapid reuptake of Ca2+.145 Ca2+‑release was therefore
considered as a signal preceding fusion, which was supported by
the analysis of vacuoles from a cmd1‑3 mutant that did release Ca2+
and was inactive in fusion.144 However, the same mutant vacuoles
were active in another study.147 Even more puzzling is the finding
that the well‑known Ca2+‑chelator BAPTA, which was thought to
inhibit fusion at docking, was bypassed if fusion was performed
at a certain ion composition, including 5 mM MgCl2,147 whereas
BAPTA is effective in inhibiting fusion if 0.5 mM MgCl2 is present
in the assay.144 Ca2+ has been implicated in other intracellular
fusion reactions, mainly defined by the sensitivity to BAPTA and
EGTA.148‑155
Can we explain the Ca2+ release independent of fusion regulation?
Potentially, the observed release of Ca2+ is due to leaky vacuoles that
do not maintain a sealed lipid bilayer throughout the fusion reaction.
It has been recently observed that vacuoles obtained from strains,
which overproduce the vacuolar SNAREs, seem to lyse partially
during fusion.215 As fusion progresses the membranes may rearrange
and seal so that the Ca2+, which was leaking out before would be
retrieved back into the vacuoles. In this scenario, Ca2+‑flux would be
a consequence of fusion, but, unlike synaptic exocytosis, independent
of an activated channel. One prediction of this model would be that
vacuoles carrying excess SNAREs also show a proportional increase
in Ca2+ efflux compared to wild‑type vacuoles. How calmodulin
then connects to vacuole fusion, will need to be addressed in future
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Glc7 also rescues MCLR‑inhibited vacuoles.181 However, MCLR
can inhibit the maturation of the reporter Pho8 within the vacuole
lumen, while still allowing fusion.36,37 Future studies therefore
need to rely on the Glc7 mutant rather than MCLR. Furthermore,
placing Glc7 into the fusion cascade will require the identification
of a substrate.
Vam10. The Vam10 protein was identified as one of the many
ORFs with a defect in vacuole morphology.158 The protein’s ORF
lies in the same chromosomal region as the VPS5 gene. Genetic and
functional studies suggest that Vam10 acts before Sec17‑Sec18‑ medi‑
ated priming, but was dispensable once priming had proceeded.51
The existence of such a reaction was implicated by earlier studies
on separate stages of the fusion reaction.44 Palmitoylation and Vps1
release were also reported to occur in a Sec17‑independent reac‑
tion,26,57 and it is possible that Vam10 is involved in one of these
subreactions.
The V1/Vo‑ATPase. The V1/Vo‑ATPase consists of a membrane‑
embedded Vo‑part and a cytosolic V1‑ATPase portion, and is
required for endosome and vacuole acidification.182 It therefore
came as a surprise, when the Vo‑part was implicated as the fusogen
of vacuole fusion;103 the large a‑subunit, Vph1, can form fusion‑
dependent homodimers late in the fusion reaction, suggesting that
Vo‑subunits form fusion pores between apposed membranes to drive
lipid bilayer mixing. In support of this finding, vph1D vacuoles are
fusion‑deficient and do not show lipid mixing,38 and antibodies
and antibody fragments to Vph1 inhibit fusion at a later time point
than antibodies to the vacuolar Q‑SNARE Vam3.101 Moreover,
biochemical experiments allowed a separation of the fusion and
pump activities of the Vo‑ATPase; the pump activity is dispens‑
able for fusion,101 but required for vacuole fission.183 These data
are consistent with observations in flies, where mutations in the
a‑subunit are associated with synaptic fusion defects at the neuro‑
muscular junction.184 Also, the exocytosis of multi‑vesicular bodies
in C. elegans, and insulin secretion in pancreatic b‑cells require the
Vo‑ATPase.185,186 The function of Vo downstream of SNAREs led to
debate, as SNAREs were considered the final fusogens and the most
likely candidates to drive lipid bilayer mixing.33,67,143,187 Indeed,
several groups have shown that SNAREs drive efficient liposome
fusion (e.g., see refs. 64 and 66). The liposome fusion assay used to
measure SNARE activity may, however, not be as robust as previ‑
ously anticipated; it requires a high SNARE density and depends
on the vesicle preparation,68,69 suggesting that additional factors
are critical for fusion in vivo. On the other hand, it was also noted
that most Vo‑deletion mutants have wild‑type‑like vacuoles;158,188
only a deletion of vph1 results in a fragmented vacuole morphology
phenotype. However, even deletions of the SNARE Nyv1, which
is clearly involved in vacuole fusion,58,71,82 result in large vacuoles,
suggesting that vacuole morphology is not always a robust phenotype
indicative of a fusion defect. It is therefore important to consider the
fusion-fission equilibrium when interpreting vacuole morphology
(see below). Based on the current knowledge of SNAREs, it is most
likely that they provide the key activity also in vacuole fusion. We
consider a mandatory Vo‑pore-mediated terminal fusion step there‑
fore less likely, whereas an involvement of the V‑ATPase in vacuole
fusion is implicated by many studies (see above). Consistent with
this statement, loss of the Vo‑subunit Vma6 on one vacuole still
results in efficient fusion with wild‑type vacuoles (ref. 183, and our
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Sec18) and becomes released during fusion progression.170 This
cycle requires Sec18, but not Sec17, is sensitive to inhibitors of
priming and to Vam3 antibodies, and is blocked in mutants in vtc1
and vtc4.104,170 Mutants lacking pbi1/ IB2 show a slight vacuole
inheritance defect and fragmented vacuoles if combined with a
sec18‑1 mutant.169,170 Interestingly, LMA1 also supports ER‑Golgi
transport, suggesting a general function in fusion.171 Since IB2 is
a cytosolic protease inhibitor, it is possible that the main role of
LMA1 is to protect the Vam3 protein (or other SNAREs) prior to its
interaction with other SNAREs in trans during docking, since some
vacuoles may lyse during fusion, and Vam3 is proteolytically sensitive
(Ungermann C, unpublished). Hence, fusion would be stimulated by
the addition of purified LMA1 compared to a standard fusion reac‑
tion where Vam3 degradation occurs. However, the defined order of
events on the vacuole for LMA1 does not exclude a more specific
role of the protein.
Mon1‑Ccz1. The peripheral membrane protein complex of Mon1
and Ccz1 has been discussed as an additional factor involved in
docking at the vacuole.73 Both proteins are conserved in eukaryotes,
and homologs have been identified in humans and C. elegans.172 In
yeast, Ccz1 was initially discovered as a protein that is functionally
linked to Ypt7.173,174 It is localized together with Mon1 to endo‑
somal and vacuolar structures, and is involved in trafficking of Prc1
and Pho8 to the vacuole.72,173,175 In addition, localization of either
Mon1 or Ccz1 depends on the other partner.72 In nematodes, the
Mon1 homolog SAND‑1 has a similar function and is involved in
late endocytic events; the depletion phenotypes of SAND‑1 and
the Ypt7‑homolog RAB7 are also very similar.172 Interestingly,
Ccz1 seems to be homologous to the Hermansky‑Pudlack protein
HPS‑4, which is involved in lysosomal trafficking in mammalian
cells,176,177 and Mon1 and Ccz1 have been proposed to contain
longin domains.178 Such a domain has been identified in several traf‑
ficking proteins, including the SNAREs Ykt6, Nyv1 and Sec22, the
s‑subunit of the AP‑complexes, and several subunits of the TRAPP
complex.179,180
So far, one study has addressed the role of Mon1 and Ccz1 in the
fusion reaction. Vacuole fusion is strongly affected if either protein
is missing or if the proteins are inhibited by specific antibodies.73
Localization of Mon1 and Ccz1 requires the class C subunits of the
HOPS complex, whereas loss of Vam6 or Vps41 leads to an increased
membrane association.73 It was therefore proposed that Mon1
and Ccz1 act at a late stage of tethering and docking, potentially
together with the HOPS complex. The dot‑like localization of the
two proteins to vacuoles, which could resemble endosomes, could
indicate specific fusion domains. Consistent with this idea, Ccz1 is
enriched at vacuole‑vacuole junctions in vivo.73 It is possible that the
Mon1‑Ccz1 complex is a cofactor of the HOPS tethering complex
in the fusion reaction.
Protein phosphatase 1/Glc7. Vacuole fusion occurs as part of the
cellular membrane dynamics during osmotic stress or inheritance.22
Like many other regulatory events, it is therefore expected to be
controlled by protein phosphorylation and dephosphorylation. At least
one kinase, the casein kinase Yck3, has been identified as a negative
regulator of fusion.71 Using immobilized phosphatase inhibitor micro‑
cystin LR (MCLR) as a bait, Mayer and colleagues identified protein
phosphatase 1 (Glc7).181 Glc7 ts‑mutants are deficient in vacuole
fusion, endocytosis and protein sorting to the vacuole. Purified
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other inhibitors in the corresponding experiments.98 The combined
data are, however, consistent with an interdependent accumula‑
tion of lipids at docking sites; ergosterol accumulation is blocked
by PI(4,5)P2 inhibitors and PI(3)P accumulation is sensitive to all
lipid‑specific ligands.98 In agreement with an active role of lipids in
fusion, lysolipid addition stabilizes the hemifusion state of docked
vacuoles and blocks fusion.195 Another quite distinct role of lipids
was suggested by mutants affecting palmitoyl‑CoA. If the synthesis
of Palmitoyl‑CoA is impaired, vacuoles fragment, most likely due to
inefficient Vac8 palmitoylation.196 Furthermore, mutants depleted
of the Palmitoyl‑CoA binding protein Acb1 also display altered
vacuole morphology,166,197 although the primary effect caused by
loss of Acb1 may be impaired sphingolipid biosynthesis. In sum, the
data strongly argue for an active role of lipids in vacuole fusion.
Protein Palmitoylation and Vacuole Fusion
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The reversible addition of palmitate to selected proteins has gained
a lot of attention over the last few years.198,199 Palmitoylation occurs
at cysteine residues on the target protein, and in general requires
a second hydrophobic segment on the protein to allow for initial
membrane contact.200 On yeast vacuoles, a number of palmitoylated
proteins were identified over the last few years, including Vac8 (see
below), the SNAREs Ykt6 and Vam3, the casein kinase Yck3, Meh1/
Ego1‑a peripheral membrane protein involved in endocytosis of the
Gap1 permease‑and the acyltransferase Pfa3 (Fig. 3).
It has been observed that palmitoylation inhibitors also block
vacuole fusion, whereas coenzyme A and palmitoyl‑CoA have a
stimulating effect.42,45,52 The most promising candidate for palmi‑
toylation during fusion turns out to be Vac8, which was identified by
Weisman and colleagues as a factor involved in vacuole inheritance
and morphology.201 Vac8 contains a myristoylated N terminus,
followed by three conserved cysteines that are targets for palmi‑
toylation and required for Vac8’s vacuole localization.202,203 Vac8 has
several functions, which seem to be defined by its membrane localiza‑
tion and the functioning of its C‑terminal armadillo repeats.74 For
inheritance, it binds via the adaptor protein Vac17 and the myosin
Myo2 to actin.204,205 It also interacts with the ER protein Nvj1 to
establish contact zones between the ER and vacuole,206 and it is
part of a complex of phosphoproteins involved in the Cvt pathway
and autophagy.21 On isolated vacuoles, Vac8 becomes palmitoylated
prior to the ATP‑dependent priming reaction.52 This reaction may
correspond to the previously identified ATP‑independent first stage
of vacuole fusion.44 It is inhibited by palmitoylation inhibitors and
antibodies to Sec18/yeast NSF and to the SNARE Ykt6, which can
stimulate Vac8 palmitoylation in vitro.52,87 Recent studies indicate
that the polytopic membrane protein of the DHHC (Asp‑His‑His‑C
ys)‑cysteine rich domain (CRD) family Pfa3, which is located on the
vacuole, provides the main palmitoylation activity for Vac8.207,208
Since Ykt6 is required in stoichiometric amounts to promote Vac8
palmitoylation in vitro,87 it is most likely not an acyltransferase but
a supporting factor. The potential direct role of Ykt6 in this process
needs further studies. As Vac8 palmitoylation occurs in a subreaction
on vacuoles, and vac8 mutants are deficient in vacuole fusion in vivo
and in vitro,42,57,195,202,207 Vac8 is thought to enhance fusion effi‑
ciency. Indeed, interaction of Vac8 with the SNARE Vam3 has been
reported, and antibodies to Vac8 block fusion at the docking stage.52
The precise molecular function of Vac8 in the fusion reaction is so
far not determined.
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unpublished observations). However, vph1D vacuoles show a strong
fusion defect when combined with wild‑type vacuoles, and fusion
cannot be rescued by the addition of recombinant Vam7.101,119,183
At present, we can only speculate, how the V‑ATPase connects to the
fusion cascade. The V‑ATPase could be directly linked to the fusion
reaction (as suggested by its association with the SNARE Vam3),103
and may be involved in the control of vacuole ion homeostasis
and lipid asymmetry, which could be required during fusion (see
above). However, this would require pump activity, which seems not
required for fusion.101 It is also possible that only the large subunit,
Vph1, which leads to vacuole fragmentation if deleted, is required
for fusion (see above).
The Vtc‑complex. A protein complex consisting of four transmem‑
brane proteins, named Vtc1 through Vtc4, was initially identified
together with the V‑ATPase and calmodulin.103 The different subunits
can be co-purified with each other and the R‑SNARE Nyv1.104
Deletions in VTC3, but not VTC1, lead to fragmented vacuoles and
a defect in biosynthetic transport to the vacuole, and all, but vtc2D
vacuoles, are fusion‑deficient.104,189 Moreover, antibodies to Vtc4
inhibit the fusion reaction in a dose‑dependent manner. The obser‑
vation that vtc1 mutants are defective in priming, and vtc3 mutant
vacuoles show Vo‑ATPase complexes, but no fusion, led to the sugges‑
tion that the Vtc proteins couple priming and fusion.104 In addition,
vacuoles lacking Vtc proteins have strongly reduced levels of LMA1
on their surface.104 Further analyses revealed that mutants deficient
in Vtc3 show decreased stability of the V‑ATPase, suggesting that
both complexes cooperate in fusion.189 Moreover, Vtc1 is genetically
connected to Cdc42 and the actin cytoskeleton.164 One weakness of
some of the above conclusions could be that most experiments on
the Vtc proteins were conducted with deletion mutants. A molecular
analysis of the interactions and functions of each Vtc‑protein may
clarify their precise function in the fusion cascade.
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Vesicle fusion and fission occur on membranes with varying lipid
compositions. It is known that lipids, which stabilize membrane
curvature at fusion or fission sites, influence the efficiency of the
respective processes.190 In addition, lipids can provide binding surfaces
for certain proteins. In vacuole fusion, evidence exists that the lipid
composition correlates with fusion efficiency and the recruitment of
fusion factors. Ergosterol is required for priming of vacuoles,99 and
an increase in ergosterol can bypass a Vrp1/Las17 requirement previ‑
ously identified for vacuole fusion.191,192 In addition, the SNARE
Vam7 binds to PI(3)P via its PX domain. Inhibiting the PI(3)‑kinase
Vps34 leads to reduced vacuole fusion and diminished Vam7 binding
to isolated vacuoles.50,91 Another phosphoinositide, PI(4,5)P2 is also
required for fusion.193,194 Ligands to PI(4,5)P2 are primarily detected
at the plasma membrane and therefore do not accumulate on vacu‑
oles in vivo.194 However, isolated vacuoles are sensitive to reagents
blocking PI(4,5)P2,193 the lipid is present at vacuole docking sites98
and it accumulates on vacuoles in mutants affecting phosphoinositide
turnover.194 Moreover, ligands to specific lipids accumulate in vitro
at vacuole docking sites as observed with fluorescence microscopy,
suggesting that PI(3)P, PI(4,5)P2, ergosterol, and diacylglycerol are
involved in organizing vacuole fusion.98 Even though such an accu‑
mulation was not observed for phosphatidylserine (PS), it should be
noted that PS is by far more abundant than, for example, PI(3)P,
and the PS‑specific inhibitor was not titrated to the same extent like
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Vacuole fusion
Figure 3. Palmitoylated proteins at the yeast vacuole. For clarity, protein transmembrane domains and the lipid bilayer are only shown schematically, while
the palmitate group is highlighted. Details are listed in the text.
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Vacuole fission is required for vacuole inheritance and in response
to osmotic stress. Several fusion and fission mutants have been
characterized that are deficient in inheritance. A complex of vacu‑
ole‑associated Vac8, the actin‑binding protein Myo2 and its adaptor
Vac17 is required for vacuole inheritance.22 Mutants lacking genes
required for inheritance often also show a defect in vacuole fragmen‑
tation. This includes several phosphoinositide remodeling proteins
such as Vac14, Vac7, and the PI(3)P kinase Fab1.29 Mutants lacking
Atg18, which binds to PI(3,5)P2, show a similar phenotype.209 Class
D proteins are required for fusion at the endosome, and class D
mutant vacuoles contain both endosomal and vacuolar markers.210
Interestingly, mutants lacking class D genes are also deficient in
inheritance and vacuole fission.26,35,71 However, the connection
between fission and fusion is not clear, since vacuole fusion may still
function normally if fission is impaired; fab1D vacuoles can still fuse
in vitro.193 Recent results indicate that acidification by the V‑ATPase
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is a prerequisite of vacuole fission;183 cells lacking V‑ATPase subunits
do not fragment if exposed to osmotic stress (ref. 183 and our
unpublished observations). The role of the V‑ATPase in this process
has not yet been determined.
The dynamin‑like protein Vps1 was the first characterized candi‑
date protein implicated in vacuole fission.26 Vps1 belongs to the class
D genes and was identified as a protein required for vesicle budding
at the TGN.211 In agreement with this, vps1D cells missort proteins
to the plasma membrane. The protein localizes mainly on dot‑like
endosomes or Golgi structures, but a fraction has also been detected
on vacuoles.26 Interestingly, vacuoles carrying no or impaired Vps1
are fusion‑deficient, and antibodies directed against Vps1 block
fusion. The release of Vps1 seems to be dependent on Vam3 and
Sec18. Mayer and colleagues suggest that Vps1 sequesters Vam3
during the fission process and is itself released prior to fusion.26 This
model is quite appealing as it connects fission and fusion through
the dynamics of a well‑characterized fission factor,210,212 but as with
every model, it will need additional experimental evidence. In vitro
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Acknowledgements
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We would like to thank Francis Barr, Karolina Peplowska,
Andreas Mayer and William Wickner for comments and discus‑
sion. This work was supported by the DFG (SFB431, UN111/5-1),
and the Fonds der Chemischen Industrie. C.U. is supported by the
Hans-Mühlenhoff foundation.
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Vacuole fusion is an integral part of vacuole biogenesis and
inheritance in yeast. The fusion machinery is required for multiple
trafficking pathways leading toward the vacuole, including autophagy
and related processes.1,17 It is therefore not surprising that such
a large number of proteins is involved in the fusion process and
vacuole morphology. Some components such as the SNARE Nyv1
are required for vacuole‑vacuole fusion,82 but not for transport to
the vacuole,86 whereas the Q‑SNAREs Vam3, Vam7 and Vti1 are
most likely required for all fusion events.83,85,213 The connection of
vacuoles to endosomes is also reflected by the recent identification
of a tethering complex at the endosome.35 The CORVET complex
shares the four class C proteins Vps11, 16, 18 and 33 with the HOPS
complex, but has the two additional subunits Vps3 and Vps8 instead
of Vps41 and Vam6. Interestingly, overexpression of Vps3 results in
the formation of an intermediate complex consisting of Vps41 and
Vps3 together with the class C proteins, which leads to fragmenta‑
tion of the vacuole. This suggests that the biogenesis of the endosome
and the vacuole is linked to the dynamics of CORVET and HOPS.
Importantly, vacuoles lacking Vps8 or Vps3 have both the endo‑
somal Rab Vps21 and Ypt7 on their membrane.35 Thus, mutants
studied for vacuole fusion may differ in their protein composition
and identity from wild‑type vacuoles.
Regulation of vacuole fusion is another important issue, which
may link fusion to trafficking. The regulatory kinase of the HOPS
complex, Yck3, is transported along the AP‑3 pathway to the
vacuole,11 and may also phosphorylate other proteins. Mutants in
Yck3 may therefore not only affect vacuole fusion, but also transport
via the AP‑3 pathway. It is likely that other trafficking processes such
as autophagy, which is upregulated by stress or starvation and directly
connected to the Tor pathway (Tor is a kinase that inhibits autophagy
under nutrient‑rich conditions), also affect vacuole fusion.17 As
kinases and phosphatases have multiple targets within the cell, a
detailed analysis of the vacuolar targets will be necessary.
sc
Connecting Vacuole Fusion to Vacuole Biogenesis
characterization. Furthermore, the link of fusion to vacuole fission is
of strong interest, and it is likely that Vps1 is only one protein among
many involved in vacuole fission. Finally, the precise mechanism
of vacuole fusion needs further studies. How does the vertex ring
fusion occur? How do lipids affect fusion? Which factor triggers the
final fusion step? The vacuole fusion system will remain an attrac‑
tive model system to unravel the molecular details underlying the
complex dynamics of biological membranes.
io
evidence for Vps1 function in vacuole fission would also strongly
support the protein’s function.
Outlook
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What are the future questions in vacuole fusion? Studies on
vacuole fusion have revealed numerous factors and proteins involved.
Future studies need to address their precise role in the fusion cascade,
and the poorly characterized factors will require particular attention.
Reconstitution of fusion, or subreactions of the fusion process, will
be one important direction. One first attempt to clarify the direct
role of fusion components was an early reconstitution approach of
vacuole fusion.214 A similar methodology using purified components
will be necessary to elucidate the precise role of each fusion factor
involved, potentially combined with SNARE reconstitution into
liposomes.66 The function of the HOPS tethering complex and its
connection to the endosomal system remains an important topic.
The multiple domains found within the HOPS subunits are likely
to connect to many proteins and reactions. Regulation of fusion
is a third important issue of interest. Only a few factors involved
in kinase cascades, such as Yck3 and Cln3, have been discussed in
this context. Other reversible modifications such as palmitoylation
are also implicated in vacuole fusion and will need further
11
Autophagy
2008; Vol. 4 Issue 1
Vacuole fusion
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