Experimental Cell Research 281, 9 –18 (2002)
doi:10.1006/excr.2002.5656
MINIREVIEW
The Central Role of the Trans-Golgi Network as a Gateway of the Early
Secretory Pathway: Physiologic vs Nonphysiologic Protein Transit
T. L. Tekirian 1
Regulation of Cellular Growth Division, Molecular Genetics, The National Cancer Institute,
Building 560, P.O. Box B, Room 22-12, Frederick, Maryland 21702
Key Words: aging; protein trafficking; Alzheimer’s disease; trans-Golgi network, TGN; endoplasmic reticulum,
ER; ubiquitin; amyloid; precursor protein; ␤-amyloid;
presenilin; Notch; furin; tau; axonal; somatodendritic;
glycosyltransferase; adaptor; vesicle; trafficking;
protein transit; protein sorting phosphoinositides.
The current review focuses upon recent advances concerning the interrelationship between the ER and the
trans-Golgi network (ER–TGN), the ER and the nucleus
(ER–nucleus), and the ER– ubiquitin–proteasomal pathways at the level of basic cell biology. The overall emphasis of this paper centers upon the high likelihood
that measurements of ER-associated protein or gene expression levels are not representative of a strict ER
alone phenotype. Rather, that ER phenotype reflects a
synthesis of phenotypes derived from intracellular compartments and phosphorylated messengers in rapport
with the ER. The ER–TGN, ER–nuclear, and ER– ubiquitin–proteasomal transit paths share the ability to feed
into the decision of whether TGN vesicles can interact
with specific phosphorylated residues in order to drive
physiologic, constitutive, anterograde traffic, retrograde traffic, and degradation. TGN vesicles can: (a) traffic to endosomes versus plasma membrane phosphodomains depending upon the presence or the absence of
select Golgi-localized gamma-ear containing ADP ribosylation factor-binding proteins and/or protein kinase D;
(b) be maintained within the TGN in the presence of a
phosphosorting acidic cluster motif adaptor; (c) transit
back to the ER via specialized TGN/ER glycosyltransferases (which modulate phosphorylated proteins); (d)
transit to the nucleus via phosphatidylinositol-4-kinaseassociated phosphodomains; and/or (e) retrotranslocate
to the ubiquitin–proteasome pathway, which is
equipped with E3 ligase potential, in order to further
regulate endosomal versus plasma membrane traffic.
The TGN is also a critical gateway for protein transit in
the sense that, as a function of sorting within this compartment, proteins are sent to the axon, cell body, or
dendrites. As the decision to sort to the axon versus the
somatodendritic compartment is intimately tied to TGN
function, future understanding of TGN biology at the
levels of neurogenesis and protein sorting is predicted
to also effectively increase our understanding of synaptic sorting/regulation. © 2002 Elsevier Science (USA)
1
SECTION I (Fig. 1)
Endoplasmic Reticulum (ER)–Intracellular Organelle
Contiguity
The stimulation of intracellular organelle cross-talk
stems largely from the endoplasmic reticulum (ER).
Hence, ER, ER-contiguous, and ER-derived compartments each serve integral roles in the overall maintenance of physiologic integrity. However, how ER–vesicular protein delivery is modulated by the molecular
phenotypes associated with ER-reciprocal compartments such as the trans-Golgi network, the nucleus,
and the ubiquitin–proteasomal system remain questions that are less well understood.
Protein transit “shuttles” have been defined simply
as membrane-bound entities (vesicles) that transit to
and fro between reciprocal intracellular organelle compartments. However, upon careful inspection of the
reciprocity that typifies intracellular organelle transport, it is apparent that trans-acting factors that modulate DNA control circuitry define the ER shuttle activities that are responsible for the modulation and
expression of intercellular and intracellular vesicles.
Both membrane-associated and membrane-independent activities enable the ER to meet the numerous
physiologic demands placed upon the whole eukaryotic
cell. Whether vesicles are guided or targeted by a
transmembrane or cytoplasmic adaptor protein, a ligand, an enzyme, or another molecule that may, for
instance, fine-tune transcriptional activity, it is the
convergence of these molecular influences that clarifies
vesicular messages.
E-mail: [email protected]. Fax: (301) 846-1666.
9
0014-4827/02 $35.00
© 2002 Elsevier Science (USA)
All rights reserved.
10
T. L. TEKIRIAN
FIG. 1. Identification of specific TGN adaptors may elucidate ties
between molecular motors involved in both early secretory transit
and axonal versus somatodendritic sorting. The feed-forward, anterograde transport of ER vesicles is associated with the routing of
proteins to the Golgi, the TGN, and/or the plasma membrane. Given
the fact that glycosyltransferases, such as GalT, can recycle between
the TGN and the ER, glycosylation may regulate nerve terminal
development as a function of recycling GalT between these compartments. The TGN itself is associated with resident adaptor activities
such as those imparted by gamma-ear containing ADP-ribosylation
factor binding proteins (GGAs), protein kinase D, and the phosphofurin–acidic cluster sorting adaptor protein termed PACS-1. GGAs
impart TGN vesicles the ability to transit to endosomes; PKD is a
core regulator of TGN to plasma membrane transport, while PACS-1
directs the localization of furin—a secreted endoprotease that catalyzes a wide array of both pathogenic and nonpathogenic proprotein
substrates. Overall, the recent identifications of these TGN adaptors
combined with the fact that the TGN is responsible for shunting
membrane proteins into selective transport vesicles destined for
axonal versus somatodendritic compartments now provides a framework with which to explore early secretory pathway regulation as
tied to axonal, cell body, dendritic, and somatodendritic sorting (Section 1).
The Trans-Golgi Network: Gateway of Physiologic
Intracellular Organelle Transit
The feed-forward, anterograde transit of vesicular
proteins within the early secretory pathway has long
been established to involve ER to Golgi and trans-Golgi
network (TGN) to plasma membrane trafficking. By
definition, the TGN is a tubulovesicular compartment
that neighbors the trans-cisterna of the Golgi and is
functionally distinct from the Golgi. The role of the
TGN as a source of exosomes that transit to the plasma
membrane, in order to provide a vesicular pool for
constitutive endocytosis, is a well accepted function of
this compartment. However, in addition to this function, recent observations support new functions that
have been accorded to this compartment, providing the
opportunity to further define the full extent of TGNmediated participation in transit regulation.
First, the TGN clearly associates with resident adaptor molecules and activities [i.e., Golgi-localized
gamma-ear containing ADP ribosylation factor binding
proteins (GGAs), protein kinase D, furin, PACS-1] that
regulate whether proteins are to be kept within this
compartment versus being shunted to the plasma
membrane for the ultimate purpose of constitutive endocytosis or targeted to the endosomal/lysosomal system for hydrolytic digestion.
Next, the TGN has recently gained attention as an
organelle that shuttles glycosyltransferases to and
from the ER. Also, the TGN appears to share transit
molecule features that have also been ascribed to the
nucleus, suggesting potential links between these two
compartments.
Last, the TGN serves as the critical relay station for
protein sorting to somatodendritic versus axonal compartments [1], wherein the decision to sort/transport a
given protein from the TGN to the axon, cell body, or
dendrite may heavily impact upon synaptic targeting.
TGN-Specific Adaptors
Whether the locale of TGN-derived vesicles serves as
a barometer of cellular trafficking versus mistrafficking is not established. We know for certain that vesicles sent from the ER through the Golgi apparatus to
the TGN can be shuttled to the plasma membrane or
the endosomal/lysosomal system. In addition, it is certainly clear that vesicles that reach the TGN can undergo retrograde transport back to the ER (i.e., as has
been classically defined for Shiga and ricin toxins [2])
or can be sent further back from the ER to the proteasome for degradative proteolysis via the process of dislocation. However, the identities of the core regulators
that selectively retain versus export TGN proteins to
the plasma membrane versus endosomes have remained unidentified for several years.
Golgi-localized gamma-ear containing ARF binding
protein (GGA) molecules have recently been isolated as
signaling molecules that impart TGN vesicles the ability to transit to endosomes (Bonafacino laboratory [3,
4]). TGN GGAs, molecules which are conserved across
yeast and humans, contain an N-terminal VHS domain
[4] that allows for phosphoinositide binding. GGA proteins are also N-terminally myristoylated, suggesting
an affinity for membrane anchorage. In addition, the
N-terminus of GGA proteins binds to a cytoplasmic tail
dileucine motif that is characteristic of transmembrane
proteins that are destined for the endosomal/lysosomal
compartment. The cloning of a dominant-negative
GGA construct that cannot be incorporated into TGN
clathrin vesicles due to the lack of a clathrin hinge
(GGAs are directly tied to clathrin recruitment and are
ARF-dependent clathrin adaptors) will now enable the
TGN AND THE EARLY SECRETORY PATHWAY
evaluation of impairment of egress from the TGN to
endosomes.
While GGA proteins appear to play a role in the
regulation of TGN vesicular egress to endosomes, protein kinase D (PKD) has recently been identified as a
molecule which critically regulates TGN vesicular
transport to the plasma membrane (Malhotra laboratory [5, 6]). PKD, a cytosolic Ser/Thr kinase interacts
with both type II PI4 and PI4P5 kinases, and its recruitment to the TGN has been proposed to require
diacylglycerol (DAG). The kinase-inactive form of PKD,
a lysine/asparagine mutant (PKD-K618N), has been
shown to localize to the TGN (colocalization with
TGN46) and imparts extensive tubulation [5]. PKDK618N containing tubules indicate a lack of detachment from the TGN and, in turn, an inhibition of cargo
transfer between the TGN and plasma membrane.
This particular PKD mutant lacks clathrin I and COPI
coats, possibly indicative of the non-clathrin-coated/
granular vesicular pool that is destined for delivery to
the cellular surface or extracellular space. Overall, this
PKD-K618N construct is completely selective for TGN
to plasma membrane transport (as protein transport
from the ER to the late Golgi, the TGN to endosomes,
and the TGN to the ER remains unaffected by its
presence). As the Bonifacino laboratory’s dominant
negative GGA construct impairs TGN vesicle egress to
endosomes and the Malhotra laboratory’s inactive
PKD precludes cargo transfer from the TGN to the
plasma membrane, it will be exciting to learn how the
effects of implementing each of these constructs in
mammalian systems will yield answers in relation to
the question of whether a specific ratio of TGN vesicles
is required for a given protein to remain in the TGN
and exit in exosomes versus degraded within mammalian cells.
In addition to PKD, furin is another molecule that
bi-cycles between the plasma membrane and the TGN
[7]. Whether furin is retained in the TGN is at least
partially modulated by the phosphofurin acidic cluster
sorting adaptor protein (PACS-1). PACS-1 directs the
localization of furin to the TGN through connecting the
furin cytoplasmic domain to clathrin adaptors and the
AP-1 sorting machinery [8]. The facts that furin can
exist within the TGN, at the plasma membrane, or
between the TGN and early endosomes when dephosphorylated by certain PP2A isotypes [9] adds complexity to the ability to discern whether pro-proteins that
are directly (i.e., serum protein pro-von Willebrand
factor; pro-nerve growth factor; Notch-1 cell surface
receptor; ECM proteins BMP-1, stromelysin-3; bacterial toxin Shiga; viral coat protein cytomegalovirus) or
indirectly affected by furin, or TGN proteins with activities that are shifted as a function of phosphorylation, are regulated by mechanisms that are inherent
within any (TGN, plasma membrane, or endosomes) or
11
all of these intracellular early secretory pathway cellular compartments.
TGN as an Organelle That Shuttles
Glycosyltransferases to and from the ER
Glycosyltransferases, enzymes that are required for
glycolipid and glycoprotein assembly, were formerly
thought to reside exclusively within the Golgi. However, the definition of glycosyltransferase localization
broadened following the execution of glycoprotein processing studies (pioneered by Nillson and co-workers;
[10, 11]) that led to the evaluation of an ER-tagged
glucosaminetransferase I that both was retained
within ER and relocated mannosidase II. Glycosyltransferases are now understood to reside in both the
ER and Golgi, suggesting that the ER produces a population of vesicles for the purpose of recycling between
these compartments. Glycosylation can also affect
phosphorylated proteins that regulate growth cone migration and protein traffic within nerve terminals.
The capacity to retrieve escaped ER proteins has
been shown to extend to the trans-most cisterna of the
Golgi stack [12, 13], as evidenced by the ability to
detect KDEL-tagged glycopeptides within the transcisterna of the Golgi. KDEL-tagged proteins were previously thought to traffic to the Golgi but not to the
TGN. However, evidence that supports TGN capture of
escaped ER residents suggests the existence of a highly
selective pathway between these two compartments.
Protein overload between the TGN and ER compartments can result in microtubule depolymerization and
galactosyltransferase (GalT) scattering [14]. The existence of shared glycosyltransferases within the TGN
and ER (i.e., GalT) suggests that these enzymes may
also serve to regulate TGN to cell surface vs TGN to
endosomal protein traffic. However, whether a pool of
TGN/ER glycosyltransferases overlaps with a TGN
pool of GGA vesicles, PKD vesicles, or furin/PACS-1
vesicles for the purpose of coordinate regulation remains unestablished.
SECTION II (Fig. 2)
The TGN, ER, and Nucleus: Potential Links
As mentioned before, there appears to be potential
for direct transit between specialized TGN–ER vesicular populations. On another level, molecules that were
formerly believed to localize strictly to the ER, nuclear
envelope, or cytosol are currently found to localize to
nuclear subdomains. A few examples of moieties which
have now been localized to the nucleolus or nucleus,
include protein disulfide isomerase (PDI), lamins, and
the translational factors eIF4A, eIF5A, eIF6, and ETF1
[15, 16]. Moreover, recent data support the localization
of phospholipids within the nucleus, rather than
12
T. L. TEKIRIAN
protein recruitment, has recently been advanced as a
ligand that stimulates PtdIns(4,5)P2 production. Of
relevance to protein transit, PtdIns(4,5)P2 is critical
for the docking of secretory vesicles and requires PI4
kinase for its synthesis. ARF1, a small GTPase that
has been implicated in vesicular coat assembly [23],
has been shown to mediate PI4 kinase recruitment and
stimulation of PtdIns(4,5)P2 on the Golgi complex [24].
Collectively, the interaction of TGN protein kinase D
with PI4 kinase, a link between the TGN and nuclear
PLD-associated PA production, and the need for PA to
stimulate PtdIns(4,5)P2 via PI4 kinase suggest that
TGN and nuclear protein transit machinery are intimately tied to phosphoinositide metabolism.
FIG. 2. Molecular parallels between the TGN and the nucleus
suggest the existence of phosphoinositide coregulation within these
organelles. To date, both the TGN and the nucleus are characterized
by the presences of (a) the consensus HEAT motif found in clathrin
adaptor and COPI coatomer subunits in the TGN and nuclear
TIP120 (a global enhancer of transcription), nuclear import protein
importin-␤, the nuclear huntingtin protein, elongation factor-3, as
well as microtubule-associated proteins; (b) phospholipase D (PLD),
a molecule that is strongly activated by the small GTPase ARF1
hydrolyzes activated phosphatidylcholine into phosphatidic acid
(PA); both PLD and PA are found selectively within the TGN and the
nucleus; (c) last, P1(4,5)P2, a molecule that docks secretory vesicles
and requires PI4 kinase for its synthesis, is situated in both the TGN
and the nucleus. The reciprocity between phosphoinositol signaling
molecules that appear to reside exclusively within the TGN and the
nucleus suggests that the TGN and nucleus, in tandem, are intimately tied to the regulation of phosphoinositol metabolism in the
context of protein transit (references within Section 2).
within the nuclear envelope [17]. In essence, the assumption that transcription and translation are spatially separated between the nucleus and cytoplasm in
eukaryotes is being rethought [18].
At least two potential ties between the TGN and the
nucleus have been raised within the literature. First,
both clathrin adaptor and COPI coatomer subunits,
key elements of vesicular assembly, contain a consensus motif (HEAT repeat) that is also found within the
TBP-associated TIP120 protein, a global enhancer of
transcription [19]. HEAT repeats have also been identified within the nuclear import protein importin-␤
[20], the nuclear huntingtin protein, elongation factor
3, as well as microtubule-associated proteins. Such
analyses [19] suggest a link between chromosome dynamics and TGN scaffolds.
Next, phospholipase D, a molecule that is activated
by the small GTPase ADP ribosylation factor 1 (ARF1)
and is responsible for the hydrolysis of phosphatidylcholine in order to generate phosphatidic acid (PA)
[21], localizes to both the trans-Golgi network and the
nucleus [22]. PA, which functions at the level of coat
Phosphoinositide Signaling Molecules and
Phosphodomains within Nuclei and the TransGolgi Network as Potential Substrates for the
Modulation of Gene Expression
In mammals, the main phosphokinase that has been
identified within the TGN is PI4 kinase (PI4K␤ (the
Saccharomyces cerevisiae equivalent of PI4K␤ is
termed Pik1 [25])). Phospholipid transfer proteins such
as sec14 (yeast) [26, 27] and PITP␤ (mammals) are
involved in PtdIns(4)P generation, while stt4 and
pik1p are Pik1 isoforms that are essential for yeast cell
growth [28, 29]. However, at this juncture, the mammalian PI4 kinase isotypes that correspond specifically
to stt4 and pik1p remain unidentified. An association
of Pik1 with the TGN has been established by virtue of
Pik1 recruitment by FRQ1 (a homologue of the mammalian neuronal calcium sensor 1 family member frequenin) [25]. FRQ1 recruitment of PI4K␤ to the Golgi
membrane and the co-immunoprecipitation of Pik1/
PI4K␤ with ARF1 suggest a role for PI4K␤/ Pik1 in the
maintenance of TGN and Golgi integrities. The fact
that TGN-localized PI4K␤/Pik1 serves as the enzyme
that allows for the formation of PtdIns(4,5)P2, combined with the observation that PtdIns(4,5)P2 localizes
to novel nuclear subdomains (phosphoinositide signaling in the nucleus has been implicated in pre-mRNA
processing [30]) may functionally tie the TGN PI4K
enzyme to the PtdIns(4,5)P2 substrate that is situated
in both the TGN and the nucleus.
Taken together, findings which support PI4K␤ coimmunoprecipitation with ARF1, PtdIns(4,5)P2 stimulation by PI4K␤ recruitment, phospholipase D recruitment by ARF1, and PLD colocalization with
PI(4,5)P2-positive nuclear subdomains suggest coordinate transit by phosphoinositide shuttles between
these compartments. The identification of shared phosphoinositide signaling molecules and phosphodomains
within the TGN and the nucleus raises the question of
whether entities such as PI4K␤ and PI(4,5)P2 exist
within these compartments for the purpose of associ-
TGN AND THE EARLY SECRETORY PATHWAY
13
ating with dynamic and transient rounds of nuclear
transcription that are coupled to given rounds of phosphorylation and dephosphorylation. Conceivably, rates
of transcriptional initiation by nuclear phosphoinositide shuttles would be tied to transcription factor turnover in that cycles of transcriptional initiation linked
to a single event of phosphorylation and dephosphorylation are not considered stably on during gene activation but dynamically reverberate between on and
off states during phosphorylation/dephosphorylation
events [31].
As an aside, the phosphatidylinositol 4-phosphate-5kinase pathway is also essential for Rac-dependent
actin assembly [32]. Given that the regulations of select G-proteins (such as Rho and Rac), phosphoinositides, and the actin cytoskeleton are central to understanding malignancies [33], future knowledge gained
in relation to how cytoplasmic versus nuclear PI kinases regulate basic transit between the nucleus, TGN,
and plasma membrane will also likely advance an understanding of cancer-associated protein trafficking.
SECTION III (Fig. 3)
A Newly Identified Function of Ubiquitin as a
Modulator of Transcription May Serve to Directly
Modulate Signal Integrity between the ER and the
Proteasome
Proteins that are destined for early secretory pathway processing undergo folding and assembly in the
ER lumen. During this process, misfolded proteins
pass through the translocon pore on the cytosolic surface of the ER and are sent through the cytosol for
dislocation to the 26S proteasome, presumably for degradation. The 26S eukaryotic proteasome ATPase complex (20S proteolytic core and two 19S regulatory complexes) has been identified as the major scavenging/
degradation pathway for a vast number of eukaryotic
nuclear and cytoplasmic proteins [34]. In higher eukaryotes, the subcellular localization of the proteasome
is mainly nuclear and cytoplasmic, whereas in yeast,
the nuclear envelope– endoplasmic reticulum network
and nuclear periphery are predominant sites of 26S
proteasome action [35].
The 26S proteasome is responsible for the breakdown of not only abnormal and damaged, misfolded
proteins, but also cell cycle regulators (mitotic regulators, G1 cyclins), tumor suppressors (c-jun, c-fos, c-mos,
E2A proteins, p53), oncogenes, antigens, and shortand long-lived secretory proteins [36]. Proteins that
are not eliminated by virtue of ubiquitin–proteasome
breakdown are degraded within the lysosomal system.
The process of conjugating ubiquitin (Ub) to lysine
residues of a target protein (ubiquitylation) involves
FIG. 3. A new role for ubiquitylation suggests a possibility for
Ub–ER–nuclear versus Ub–ER–proteosomal pools. As determined by
the fact that Ub-mediated proteolysis is not required for transcriptional activation, the process of ubiquitylation itself has been ascribed a new function—that of a transcriptional modulator [43]. This
finding raises the question of whether two distinct, non-overlapping
vesicular pools play a role in the cell’s decision to (a) modulate
ER–nuclear gene expression versus (b) dislocate a given protein from
the ER to cytosol to the proteasome for degradation (degradative
proteolysis). How a Ub–ER–nuclear shuttle versus a Ub–proteasomal shuttle may differentially regulate ER/TGN/plasma membrane/
endosomal signaling is a newly emerging field of study. However, it
is already clear that Ub ligase (i.e., c-cbl) can modulate plasma
membrane versus endocytic sorting [44]—thus providing preliminary evidence for ER ubiquitylation, ER–TGN– endosomal versus
ER–TGN–plasma membrane intracellular sorting rapports (Section
3).
Ub-activating enzymes (termed E1 enzymes), Ub-conjugating enzymes (E2), and Ub-ligating enzymes (E3).
E3 ligases recognize a given degradation signal (a degron) within an acceptor protein and serve as potent
regulators of ubiquitin timing and substrate selection.
Salghetti and colleagues [37] have observed the fact
that sequences that activate transcription [transcriptional activation domains (TADs)] and signal degradation (“degrons”) functionally overlap. Such overlap exists, for instance, in the cases of myc, jun, fos, myb,
␤-catenin, HIF-1␣, E2F-1, p53, rel, and GCN-4 sequences). The presence of Ub has been equated with
disease-associated phenotypes based upon its established ability to tag and target proteins for proteasomal
proteolysis. A few examples of ubiquitin ties to neuropathologic conditions include Ub-positive polyglu-
14
T. L. TEKIRIAN
FIG. 4. The TGN serves as a core modulator of Alzheimer’s disease molecular protein transit. A compilation of evidence supporting a
central role for the TGN in the course of AD pathophysiology is provided in Table 1 and depicted above. These lines of evidence, combined
with new knowledge concerning TGN adaptor function relative to those functions imparted by contiguous early secretory pathway transit
sites (Figs. 1–3) suggest that (a) the regulation of APP via the ␥-secretase action of presenilin(s) likely commands a need to understand
whether furin is cleaved within the TGN versus the plasma membrane (whether these decisions are modulated by PACS-1, GGAs, or PKD
in the case of PS and APP is yet to be determined). (b) Titration of TGN glycosylation of PS-binding partners that prove essential for PS
activity is predicted to ultimately regulate the equilibrium of proper axonal (i.e., tau) vs somatodendritic (microtubule-associated protein)
sorting. (c) Last, recycling of AD-associated proteins that are found in both the TGN and the ER may very well be tied to phosphorylationdependent cycles of on– off transcription that are modulated by TGN/nuclear-selective phosphoproteins, TGN/nuclear phosphorylationdependent activities (i.e., those imparted by PLD, PI4K-␤ and PI4,5P2), and shared TGN and nuclear (i.e., HEAT) motifs (Section 3).
tamine protein inclusions [38], huntingtin protein inclusions [39], ubiquitin-positive Alzheimer’s disease
presenilin protein oligomers [40, 41], and the association of autosomal-recessive juvenile parkinsonism with
a causative gene that encodes an E3 ligase [42]. While
the ubiquitin–proteasome pathway is the major degradative pathway for most proteins, why or whether the
presence of ubiquitin would be linked exclusively to
degenerative phenotypes has remained unclear.
Quite recently [43], ubiquitin has been ascribed a
new function, one which supports a function for Ub as
a transcriptional modulator. Specifically, Salghetti and
colleagues assessed how ubiquitylation regulates transcription factor activity. These investigators evaluated
a known herpes simplex virus (VP16) TAD degron
fused to a Lex-A DNA binding domain in contrast to
myc and yeast G1 cyclin Cln3 TAD degrons (each also
fused to Lex-A binding domains). The results of this
study indicate that a Met 30 residue is required for
VP16 transactivation and that the evaluated VP16 –
Lex-A fusion (but not the myc or Cln3 fusions) was
unable to activate transcription in the absence of Met
30. These data indicate that ubiquitylation is required
for VP16 transactivator function and reveal that the
degron function of the VP16 TAD is intimately tied to
an ability to activate transcription. Overall, this work
demonstrates that Ub-mediated proteolysis is not required for transcriptional activation.
This newly ascribed function of Ub as a transcriptional activator, in the absence of Ub-mediated proteolysis, raises the question of whether the appropriate
recruitments of Ub-activating, -conjugating, and -ligat-
TGN AND THE EARLY SECRETORY PATHWAY
15
TABLE 1
Central Connections between Alzheimer’s Disease Molecules and Trans-Golgi Network (TGN)
Protein Localization, Modification, or Transport
1. Furin localizes in the TGN [48, 49].
2. Requirement of furin to activate ␤-site amyloid precursor protein-cleaving enzyme (BACE) [50–52].
3. BACE1 localizes to the TGN and binds to nicastrin (a molecule which interacts with PS and is required in order to impart ␥secretase activity) [53].
4. Cofractionation of PS N- and C-terminal fragments with the trans-Golgi network enzyme galactosyltransferase [54, 55].
5. PS1 and PS2 interact with Rab 11 (a TGN-associated G protein) [56]. PS1 is a membrane receptor for Rab GDI, part of the GTPase
molecular switch apparatus that regulates vesicular transport (GDI extracts GDP-bound rab from membrane and deposits it into
cytoplasm) [57].
6. Co-immunoprecipitation of clathrin-coated vesicles [CCVs] with adaptin and PS1 [58].
7. Localization of mature APP and APP C-terminal fragments with CCVs [59].
8. NPX Y-targeting motif within APP, which supports its transit to the TGN [60].
9. Competition for ␣- and ␤-secretase cleavages of APP, within the TGN [61].
10. APP destined for ␤-secretase processing is sorted into a distinct trans-Golgi or endosomal compartment prior to transport that is
mediated by rab 6 (G-protein localized to post-Golgi, trans-Golgi cisternae, and TGN) [62].
11. Localization of highly abundant ␤-amyloid species to the TGN [63–67].
12. Estrogen reduces A␤ generation through the stimulation of TGN–vesicle biogenesis [67].
ing enzymes are tied to transcriptional modulation versus degradative proteolysis in the context of regulating
signal integrity in protein traffic. A role for ubiquitin as
a transcriptional regulator also raises the issue of
whether there are two distinct ER– ubiquitin vesicle
pools. One vesicular pool could conceivably play a role
in the cell’s decision to dislocate a given (mammalian)
protein from the ER through cytosol to the proteasome
for degradation in contrast to a separate pool that
selectively shuttles molecules between the ER and the
nucleus in order to modify gene expression.
The new link of ubiquitin to transcriptional activation raises the question of whether Ub pathway components ascribed to the degradative, Ub-mediated proteolysis versus ubiquitin-mediated transcriptional
activation pathways differentially control nuclear, ER/
TGN/plasma membrane signaling decisions. Recently,
a specific E3– ubiquitin ligase, c-cbl, has been associated with an ability to ubiquitinate the EGF receptor
at the plasma membrane and remains associated with
the receptor throughout the clathrin-mediated endocytic pathway [44]. The role of c-cbl as a Ub ligase that
modulates endocytic versus plasma membrane sorting
further supports the concept that ER– ubiquitin, ER–
TGN– endosomal, and ER–TGN–plasma membrane intracellular sorting rapports are indeed linked.
The ability of the ER to respond to a variety of
insults [whether responding to hypoxia, hypoglycemia, protein aggregation (insolubility, misfolding),
or overexpression] is clearly tied to a capability to
adapt to secretory apparatus demand. In the context
of normal development, ER-responsive pathways
modulate gene expression levels and protein folding
machinery in order to impart physiologic conformational protein folding. Physiologic protein folding has
been equated with fluid, feed-forward anterograde
transport that is not associated with misproteolysis
or nonphysiologic, aberrant, degradation signals.
However, cell routes that have previously been considered ER stress paths are now recognized as
shared with signaling paths that are implicated in
normal development (i.e., in the case of apoptosis). In
light of a propensity for ER-responsive pathways to
be connected with both normal developmental and
misfolded protein response, it is not a stretch of the
imagination to add Ub tagging to the repertoire of
modulators of both normal developmental transcriptional regulation and proteolytic degradation.
For years, the pathophysiologies of disorders that
are typified by protein aggregation (for instance,
Huntington’s disease, familial Parkinson’s disease,
lipofuscinosis, macular degeneration, Alzheimer’s
disease) have been equated with the concept that
extracellular protein aggregates serve as a focal initiator of disease. However, it is now apparent that
inter- and intracellular organelle transit and vesicular constitution (rather than simply the extrusion
of protein into the extracellular matrix) gauge the
eukaryotic cell’s decision to regulate whether a given
protein or lipid is shuttled through traditional, constitutive transport versus degraded. Whether the
regulation of functional protein trafficking versus
mistrafficking is regulated mainly by (a) phosphoinositide signal transduction, (b) phospholipid composition, (c) organelle vesicular phosphodomain function,
(d) phosphoadaptor proteins, (e) affinity versus avidity of binding between vesicles that oscillate between
these various phosphorylated entities, or (f) a capacity for phosphoinositides themselves to serve as signals that shape whether cell surface receptors will be
further modified remains to be seen.
16
T. L. TEKIRIAN
Alzheimer’s Disease: Molecular Genetics, APP, and
The Presenilins: Connectivity of Alzheimer’s Disease
Molecules/Molecular Processes and Trans-Golgi
Network Protein Localization, Modification, and/or
Transport
Studies of individuals who are affected by familial
(inherited) Alzheimer’s disease (FAD) provide essential genetic clues that enhance the understanding of
both inherited and sporadic (presumably noninherited)
forms of this disorder. Three genes serve as critical
regulators of inherited AD pathophysiology. One of
these genes encodes the amyloid precursor protein
(APP), another encodes the polytopic, seven-transmembrane spanning presenilin 1 protein (PS1), and a
third encodes the presenilin 2 protein (PS2; PS2 is 67%
identical to the PS1 sequence at the amino acid level)
(for reviews, please refer to [41, 45]).
According to co-immunoprecipitation studies, APP
and the presenilins (PS) interact with one another [46].
While the function of APP remains elusive, presenilin
has recently been ascribed an enzymatic activity
termed “gamma (␥-) secretase activity.” ␥-Secretase
activity [47] liberates the C-terminus of the ␤-amyloid
peptide that is typically sequestered within APP (in
the absence of ␥-secretase cleavage) and (either directly or indirectly) imparts an elevation in ␤-amyloid
x-42 (A␤x-42) peptide levels. Increased levels of A␤x-42
protein are a common neuropathologic hallmark
within brains and cerebrospinal fluid of those affected
by either sporadic or familial AD. Another hallmark
type of AD pathology is the “neurofibrillary tangle,” an
intracellular deposit that is composed of axonal tau
protein (“paired helical filaments”) and dystrophic neurites (neuritic debris). How and whether the genesis of
A␤x-42 (deposition begins in higher cortical regions)
within AD brain is tied to tau protein genesis (deposition beings in the entorhinal cortex) remains unclear.
Each APP and the presenilins undergo proteolysis.
While it is clear that each of these molecules is subject
to multiple cleavages, the physiologic relevance of such
cleavage events remains unelucidated. APP undergoes
maturation within the early secretory pathway, is subject to posttranslational modifications within the ER
and trans-Golgi compartments, and is thought to undergo constitutive recycling at the cell surface. While a
physiologic explanation for PS endoproteolysis is as yet
unestablished, furin has been shown to regulate both
PS and Notch (a molecule that serves a critical role in
the developmental regulation of cell fate) activities.
Alzheimer’s disease-associated questions that directly
relate to the function of the TGN, which remain unaddressed experimentally, include (1) what are the subcellular regulators of mechanisms that account for
whether or not presenilin is cleaved by furin within the
TGN vs at the plasma membrane? It may be possible
that the presenilins may sometimes reside at the cell
surface and, at other times may not, depending upon
whether furin resides within the TGN versus at the
plasma membrane, endosomes or potentially, ubiquitin-ligase containing vesicles that may facilitate
ectodomain-shedding and degradation. (2) How do specific TGN adaptor proteins modulate the ␥-secretase
activity complex components PS and nicastrin? The
modulation of a balance between glycosylation of APP
and presenilin-interacting proteins (and resultant constitutive plasma membrane processing) in contrast to
the decision to degrade the presenilins/presenilin-interacting proteins through a TGN to ER to proteasomal
path may be one mode through which ␥-secretase activity is regulated. (3) How do the regulations of furin
and ␥-secretase activity within the trans-Golgi network vs the plasma membrane impact the genesis of
specific ␤-amyloid and tau isoforms? The possibility
that TGN sorting to the somatodendritic versus axonal
compartments titrates the degree of intracellular tau
versus intra- versus extracellular A␤ manufactured by
a given cell is certainly one possibility. As we carefully
digest sapient contributions as ebb tides imparted by
those who study protein transit and disease, we draw
nearer to threading the many puzzles once thought
intangible.
Many kind thanks to all of the members of the Genetics and Aging
Unit, Massachusetts General Hospital, Harvard Medical School,
1998 –2001, who collectively provided the intellectual atmosphere
which gave rise to my interest in the field of protein transit.
REFERENCES
1.
Trimmer, J. S. (1999). Sorting out receptor trafficking. Neuron
22, 411– 412.
2.
Johannes, L., and Goud, B. (1998). Surfing on a retrograde
wave: how does shiga toxin reach the ER? Trends Cell Biol. 8(4),
158 –162.
3.
Mullens, C., and Bonifacino, J. S. (2001). Structural requirements for function of yeast GGAs in vacuolar protein sorting,
alpha-factor maturation, and interactions with clathrin. Mol.
Cell Biol. 21(23), 7981–7994.
4.
Misra, S., Puertollano, R., Kato, Y., Bonifacino, J. S., and Hurley, J. H. (2002). Structural basis for acidic-cluster dileucine
sorting-signal recognition by VHS domains. Nature 415, 933–
935.
5.
Liljedahl, M., Maeda, Y., Colanzi, A., Ayala, I., Van Lint, J., and
Malhotra, V. (2001). Protein Kinase D regulates the fission of
cell surface destined transport carriers from the trans-Golgi
Network. Cell 104, 409 – 420.
6.
Baron, C. L., and Malhotra, V. (2002). Role of diacylglycerol in
PKD recruitment to the TGN and protein transport to the
plasma membrane. Science 295, 325–328.
7.
Molloy, S. S., Anderson, E. D., Jean, F., and Thomas, G. (1999).
Bicycling the furin pathway: from TGN localization to pathogen
activation and embryogenesis. Trends Cell Biol. 9(1), 28 –35.
8.
Wan, L., Molloy, S. S., Thomas, L., Liu, G., Xiang, Y., and
Rybak, S. L. (1998). PACS-1 defines a novel gene family of
TGN AND THE EARLY SECRETORY PATHWAY
cytosolic sorting proteins required for TGN localization. Cell 94,
205–216.
9.
10.
11.
Jones, B. G., Thomas, L., Molloy, S. S., Thulin, D. D., Fry, M. D.,
Walsh, K. A., and Thomas, G. (1995). Intracellular trafficking of
furin is modulated by the phosphorylation state of casein kinase
II in its cytoplasmic tail. EMBO J. 14, 5869 –5883.
Nilsson, T., Pypaert, M., Hoe, M. H., Slusarewicz, P., Berger,
E. G., and Warren, G. (1993). Overlapping distribution of two
glycosyltransferases in the Golgi apparatus of HeLa cells.
J. Cell Biol. 120(1), 5–13.
Nilsson, T., Slusarewicz, P., Hoe, M. H., and Warren, G. (1994).
Kin recognition. A model for the retention of Golgi enzymes.
FEBS Lett. 330(1), 1– 4.
12.
Miesenbock, G., and Rothman, J. E. (1995). The capacity to
retrieve escaped ER proteins extends to the trans-most cisterna
of the Golgi Stack. J. Cell Biol. 129(2), 309 –319.
13.
Griffiths, G., Ericsson, M., Krinjnse-Locker, J., Nilsson, T.,
Goud, B., Soeling, H. D., Tang, B. L., Wong, S. H., and Hong, W.
(1994). Localization of the Lys, Asp, Glu, Leu tetrapeptide
receptor to the Golgi complex and the intermediate compartment in mammalian cells. J. Cell Biol. 127, 1557–1574.
14.
Storrie, B., White, J., Rottger, S., Stelzer, E. H., Suganuma, T.,
and Nilsson, T. (1998). Recycling of golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering.
J. Cell Biol. 143(6), 1505–1521.
17
26.
Franzusoff, A., Redding, K., Crosby, J., Fuller, R. S., and Schekman, R. (1991). Localization of components involved in protein
transport and processing through the yeast Golgi apparatus,
J. Cell Biol. 112, 27–37.
27.
Tschopp, J., Esmon, P. C., and Schekman, R. (1984). Defective
plasma membrane assembly in yeast secretory mutants. J.
Bacteriol. 160(3), 966 –970.
28.
Flanagan, C. A., Schnieders, E. A., Emerick, A. W., Kunisawa,
R., Admon, A., and Thorner, J. (1993). PI4-kinase: Gene structure and requirement for yeast cell viability. Science 262, 1444 –
1448.
29.
Audhya, A., Foti, M., and Emr, S. D. (2000). Distinct roles for
the yeast PI4 kinases, Stt4p and Pik1p, in secretion, cell
growth, and organelle membrane dynamics. Mol. Biol. Cell. 11,
2673–2689.
30.
Boronenkov, I. V., Loijens, J. C., Umeda, M., and Anderson,
R. A. (1998). Phosphoinositide signaling pathways in nuclei are
associated with nuclear speckles containing pre-mRNA processing factors. Mol. Biol. Cell. 9, 3547–3560.
31.
Hazzalin, C. A., and Mahadevan, L. C. (2002). MAPK-regulated
transcription: A continuously variable gene switch? Nat. Rev.
Mol. Cell Biol. 3, 30 –37.
32.
Tolias, K. F., Hartwig, H. H., Ishihara, H., Shibasaki, Y., Cantley, L. C., and Carpenter, C. L. (2000). Type I alpha PI4 phosphate 5 kinase mediates Rac-dependent actin assembly. Curr.
Biol. 10(3), 153–156.
33.
Maruta, H., He, H., Tikoo, A., Vuong, T., and Nur-E-Kamal, M.
(1999). G proteins, phosphoinositides, and actin-cytoskeleton in
the control of cancer growth. Microsc. Res. Tech. 47(1), 61– 66.
34.
Zwickl, P., Voges, D., and Baumeister, W. (1999). The proteasome: A macromolecular assembly designed for controlled proteolysis. Philos. Trans R. Soc. London 354, 1501–1511.
35.
Schauer, T. M., Nesper, M., Kehl, M., Lottspeich, F., MullerTaubenberger, A., Gerisch, G., and Baumeister, W. (1993). Proteasomes from Dictyostelium discoideum: Characterization of
structure and function. J. Struct. Biol. 111, 135–147.
15.
Andersen, J. S., Kyon, C. E., Fox, A. H., Keung, A. K. L., Lam,
Y. W., Steen, H., Mann, M., and Lamond, A. I. (2002). Directed
proteomic analysis of the human nucleolus. Curr. Biol. 12,
1–11.
16.
Dundr, M., and Misteli, T. (2002). Nucleolomics: An inventory
of the nucleolus. Mol. Cell 9(1), 5– 6.
17.
Irvine, R. (2000). Nuclear lipid signaling. Science STKE, RE1–
12.
18.
Iborra, F. J., Jackson, D. A., and Cook, P. R., (2001). Coupled
transcription and translation within nuclei of mammalian cells.
Science 293, 1139 –1142.
36.
19.
Neuwald, A. F., and Hirano, T. (2000). HEAT repeats associated with condensins, cohesins, and other complexes involved
in chromosome-related functions. Genome Res. 10, 1445–1452.
Lee, D. H., and Goldberg, A. L. (1998). Proteasome inhibitors:
Valuable new tools for cell biologists. Trends Cell Biol. 8, 397–
403.
37.
20.
Lee, S. J., Imamoto, N., Sakai, H., Nakagawa, A., Kose, S.,
Koike, M., Yamamoto, M., Kumasaka, T., Yoneda, Y., and
Tsukihara, T. (2000). The adoption of a twisted structure of
importin-beta is essential for protein-protein interaction required for nuclear transport. J. Mol. Biol. 302, 251–264.
Salghetti, S. E., Muratani, M., Wijnen, H., Futcher, B., and
Tansey, W. P. (2000). Functional overlap of sequences that
activate transcription and signal ubiquitin-mediated proteolysis. 97(7), 3118 –3123.
38.
Schmidt, T., Lindenberg, K. S., Krebs, A., Schols, L., Laccone,
F., Herms, J., Rechsteiner, M., Riess, O., and Landwehrmeyer,
G. B. (2002). Protein surveillance machinery in brains with
spinocerebellar ataxia type 3: Redistribution and differential
recruitment of 26S proteasome subunits and chaperones to
neuronal intranuclear inclusions. Ann. Neurol. 51(3), 302–310.
39.
Martin-Aparicio, E., Yamamoto, A., Hernandez, F., Hen, R.,
Avila, J., and Lucas, J. J. (2001). Proteasomal-dependent aggregate reversal and absence of cell death in a conditional
mouse model of Huntington’s disease. J. Neurosci. 21(22),
8772– 8781.
40.
Kim, T. W., Pettingell, W. H., Hallmar, O. G., Moir, R. D.,
Wasco, W., and Tanzi, R. E. (1997). Endoproteolytic cleavage
and proteasomal degradation of presenilin 2 in transfected
cells. J. Biol. Chem. 272(17), 11006 –11010.
41.
Kim, T. W., and Tanzi, R. E. (1997). Presenilins and Alzheimer’s disease. Curr. Opin. Neurobiol. 7(5), 683– 688 [Review].
42.
Tanaka, K., Suzuki, T., Chiba, T., Shimura, H., Hattori, N., and
Mizuno, Y. (2001). Parkin is linked to the ubiquitin pathway. J.
Mol. Med. 79(9), 482– 494.
21.
Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C.,
and Roth, M. G. (1996). Evidence that PLD mediates ARFdependent formation of Golgi-coated vesicles. J. Cell Biol. 134,
295–306.
22.
Freyburg, Z., Sweeney, D., Siddhanta, A., Bourgoin, S., Frohman, M., and Shields, D. (2001). Intracellular localization of
PLD1 in mammalian cells. Mol. Biol. Cell 12, 943–955.
23.
Goldberg, J. (1999). Structural and functional analysis of the
ARF1-ARFGAP complex reveals a role for coatomer in GTP
hydrolysis. Cell 96(6), 893–902.
24.
Godi, A., Pertile, P., Meyers, R., Marra, P., Di Tullio, G.,
Iuriscci, C., Luini, A., Corda, D., and De Matteis, M. A. (1999).
ARF mediates recruitment of PtdIns-4-OH kinase-␤ and stimulates synthesis of PtdIns (4,5)P2 on the Golgi complex. Nat.
Cell Biol. 1, 280 –287.
25.
Walch-Solimena, C., and Novick, P. (1999). The yeast PI-4-OH
kinase Pik1 regulates secretion at the Golgi. Nat. Cell Biol. 1(8),
523–525.
18
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
T. L. TEKIRIAN
Salghetti, S. E., Caudy, A. A., Chenowetch, J. G., and Tansey,
W. P. (2001). Regulation of transcriptional activation domain
function by ubiquitin. Science 293, 1651–1653.
de Melker, A. A., van der Horst, G., Calafat, J., Jansen, H., and
Borst, J. (2001). c-Cbl ubiquitinates the EGF receptor at the
plasma membrane and remains receptor associated throughout
the endocytic route. J. Cell Sci. 114, 2167–2178.
Tanzi, R. E., Kovacs, D. M., Kim, T. W., Moir, R. D., Guenette,
S. Y., and Wasco, W. (1996). The gene defects responsible for
familial Alzheimer’s disease. Neurobiol. Dis. 3(3), 159 –168 [Review].
Weidemann, A., Paliga, K., Durrwang, U., Czech, C., Evin, G.,
Masters, C. L., and Beyreuther, K. (1997). Formation of stable
complexes between two Alzheimer’s disease gene products: Presenilin-2 and beta-amyloid precursor protein. Nature Med. 3(3),
328 –332.
Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly,
W. T., and Selkoe, D. J. (1999). Two transmembrane aspartates
in presenilin-1 required for presenilin endoproteolysis and
gamma-secretase activity. Nature 398(6727), 513–517.
Teuchert, M., Schafer, W., Berghofer, S., Hoflack, B., Klenk,
H. D., and Garten, W. (1999). Sorting of furin at the trans-Golgi
network. Interaction of the cytoplasmic tail sorting signals with
AP-1 Golgi-specific assembly proteins. J. Biol. Chem. 274(12),
8199 – 8207.
Band, A. M., Maatta, J., Kaariainen, L., and Kuismanen, E.
(2001). Inhibition of the membrane fusion machinery prevents
exist from the TGN and proteolytic processing by furin. FEBS
Lett. 505(1), 118 –124.
Bennett, B. D., Denis, P., Haniu, M., Teplow, D. B., Kahn, S.,
Louis, J. C., Citron, M., and Vassar, R. (2000). A furin-like
convertase mediates propeptide cleavage of BACE, the Alzheimer’s beta-secretase. J. Biol. Chem. 275(48), 37712–37717.
Creemers, J. W., Ines Dominguez, D., Plets, E., Serneels, L.,
Taylor, N. A., Multhaup, G., Craessaerts, K., Annaert, W., and
De Strooper, B. (2001). Processing of beta-secretase by furin
and other members of the proprotein convertase family. J. Biol.
Chem. 276(6), 4211– 4217.
Benjannet, S., Elagoz, A., Wickham, L., Mamarbachi, M., Munzer, J. S., Basak, A., Lazure, C., Cromlish, J. A., Sisodia, S.,
Checler, F., Chretien, M., and Seidah, N. G. (2001). Post-translational processing of beta-secretase (beta-amyloid-converting
enzyme) and its ectodomain shedding. The pro- and transmembrane/cytosolic domains affect its cellular activity and amyloidbeta production. J. Biol. Chem. 276(14), 10879 –10887.
Hattori, C., Asai, M., Oma, Y., Kino, Y., Sasagawa, N., Saido,
T. C., Maruyama, K., and Ishiura, S. (2002). BACE1 interacts
with nicastrin. Biochem. Biophys. Res. Commun. 293(4), 1228 –
1232.
Xia, W., Zhang, J., Ostaszewski, B. L., Kimberly, W. T.,
Seubert, P., Koo, E. H., Shen, J., and Selkoe, D. J. (1998).
Presenilin 1 regulates the processing of beta-amyloid precursor
protein C-terminal fragments and the generation of amyloid
beta-protein in endoplasmic reticulum and Golgi. Biochemistry
37(47), 16465–16471.
Zhang, J., Kang, D. E., Xia, W., Okochi, M., Mori, H., Selkoe,
D. J., and Koo, E. H. (1998). Subcellular distribution and turn-
Received August 9, 2002
Published online October 11, 2002
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
over of presenilins in transfected cells. J. Biol. Chem. 273(20),
12436 –12442.
Dumanchin, C., Czech, C., Campion, D., Cuif, M. H., Poyot, T.,
Martin, C., Charbonnier, F., Goud, B., Pradier, L., and Frebourg, T. (1999). Presenilins interact with Rab11, a small GTPase involved in the regulation of vesicular transport. Hum.
Mol. Genet. 7, 1263–1269.
Scheper, W., Zwart, R., Sluijs, P., Annaert, W., Gool, W. A., and
Baas, F. (2000). Alzheimer’s presenilin 1 is a putative membrane receptor for rab GDP dissociation inhibitor. Hum. Mol.
Genet. 9(2), 303–310.
Efthimiopoulos, S., Floor, E., Georgakopoulos, A., Shioi, J., Cui,
W., Yasothornsrikul, S., Hook, V. Y., Wisniewski, T., Buee, L.,
and Robakis, N. K. (1998). Enrichment of presenilin 1 peptides
in neuronal large dense-core and somatodendritic clathrincoated vesicles. J. Neurochem. 71(6), 2365–2372.
Nordstedt, C., Caporaso, G. L., Thyberg, J., Gandy, S. E., and
Greengard, P. (1993). Identification of the Alzheimer beta/A4
amyloid precursor protein in clathrin-coated vesicles purified
from PC12 cells. J. Biol. Chem. 268(1), 608 – 612.
Borg, J. P., Ooi, J., Levy, E., and Margolis, B. (1996). The
phosphotyrosine interaction domains of X11 and FE65 bind to
distinct sites on the YENPTY motif of amyloid precursor protein. Mol. Cell Biol. 16(11), 6229 – 6241.
Skovronsky, D. M., Moore, D. B., Milla, M. E., Doms, R. W., and
Lee, V. M. (2000). Protein kinase C-dependent alpha-secretase
competes with beta-secretase for cleavage of amyloid-precursor
protein in the trans-golgi network. J. Biol. Chem. 275(4), 2568 –
2575.
McConlogue, L., Castellano, F., deWit, C., Schenk, D., and
Maltese, W. A. (1996). Differential effects of a Rab6 mutant on
secretory versus amyloidogenic processing of Alzheimer’s betaamyloid precursor protein. J. Biol. Chem. 271(3), 1343–1348.
Xu, H., Sweeney, D., Greengard, P., and Gandy, S. (1996).
Metabolism of Alzheimer beta-amyloid precursor protein: Regulation by protein kinase A in intact cells and in a cell-free
system. Proc. Natl. Acad. Sci. USA 93(9), 4081– 4084.
Xu, H., Sweeney, D., Wang, R., Thinakaran, G., Lo, A. C.,
Sisodia, S. S., Greengard, P., and Gandy, S. (1997). Generation
of Alzheimer beta-amyloid protein in the trans-Golgi network
in the apparent absence of vesicle formation. Proc. Natl. Acad.
Sci. USA 94(8), 3748 –3752.
Hartmann, T., Bieger, S. C., Bruhl, B., Tienari, P. J., Ida, N.,
Allsop, D., Roberts, G. W., Masters, C. L., Dotti, C. G., Unsicker,
K., and Beyreuther, K. (1997). Distinct sites of intracellular
production for Alzheimer’s disease A beta40/42 amyloid peptides. Nature Med. 3(9), 1016 –1020.
Greenfield, J. P., Tsai, J., Gouras, G. K., Hai, B., Thinakaran,
G., Checler, F., Sisodia, S. S., Greengard, P., and Xu, H. (1999).
Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer beta-amyloid peptides. Proc.
Natl. Acad. Sci. USA 96(2), 742–747.
Greenfield, J. P., Leung, L. W., Cai, D., Kaasik, K., Gross, R. S.,
Rodriguez-Boulan, E., Greengard, P., and Xu, H. (2002). Estrogen lowers Alzheimer beta-amyloid generation by stimulating
trans-Golgi network vesicle biogenesis. J. Biol. Chem. 277(14),
12128 –12136.