A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network

TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell Biol. 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, approximately 90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP- binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was approximately 250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was approximately 86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN.     

Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a posttranslational modification occurring in the endoplasmic reticulum (ER). After GPI attachment, proteins are transported by coat protein complex II (COPII)-coated vesicles from the ER. Because GPI-anchored proteins (GPI-APs) are localized in the lumen, they cannot interact with cytosolic COPII components directly. Receptors that link GPI-APs to COPII are thought to be involved in efficient packaging of GPI-APs into vesicles; however, mechanisms of GPI-AP sorting are not well understood. Here we describe two remodeling reactions for GPI anchors, mediated by PGAP1 and PGAP5, which were required for sorting of GPI-APs to ER exit sites. The p24 family of proteins recognized the remodeled GPI-APs and sorted them into COPII vesicles. Association of p24 proteins with GPI-APs was pH dependent, which suggests that they bind in the ER and dissociate in post-ER acidic compartments. Our results indicate that p24 complexes act as cargo receptors for correctly remodeled GPI-APs to be sorted into COPII vesicles.     

Rab1 and Ca2+ are required for the fusion of carrier vesicles mediating endoplasmic reticulum to Golgi transport

Members of the rab/YPT1/SEC4 gene family of small molecular weight GTPases play key roles in the regulation of vesicular traffic between compartments of the exocytic pathway. Using immunoelectron microscopy, we demonstrate that a dominant negative rab1a mutant, rab1a(N124I), defective for guanine nucleotide binding in vitro, leads to the accumulation of vesicular stomatitis virus glycoprotein (VSV-G) in numerous pre-cis-Golgi vesicles and vesicular-tubular clusters containing rab1 and beta-COP, a subunit of the coatomer complex. Similar to previous observations (Balch et al. 1994. Cell. 76:841-852), VSV-G was concentrated nearly 5-10-fold in vesicular carriers that accumulate in the presence of the rab1a(N124I) mutant. VSV-G containing vesicles and vesicular-tubular clusters were also found to accumulate in the presence of a rab1a effector domain peptide mimetic that inhibits endoplasmic reticulum to Golgi transport, as well as in the absence of Ca2+. These results suggest that the combined action of a Ca(2+)-dependent protein and conformational changes associated with the GTPase cycle of rab1 are essential for a late targeting/fusion step controlling the delivery of vesicles to Golgi compartments.     

Multicompartmental distribution of the tuberous sclerosis gene products,hamartin and tuberin

Mutations of the TSC1 and TSC2 genes give rise to the clinical disorder of tuberous sclerosis characterized by the development of hamartomas predominantly affecting the central nervous system, kidney, skin, lung, and heart. The function of the gene products, hamartin and tuberin, is not well understood but we have previously suggested a role in vesicular transport. To define the subcellular compartment(s) involved with these two proteins, biochemical characterization of hamartin and tuberin was performed in primary tissues and cell lines. Fractionation of cell lysates identified both proteins in the cytosolic, microsomal, and cytoskeletal compartments. In each of these fractions, hamartin and tuberin formed a stable complex in coimmunoprecipitation analyses. Further, they colocalized extensively in discrete, vesicular structures in the cytoplasm. Within the microsomal compartment, hamartin and tuberin behaved as peripheral membrane proteins that associate with the cytosolic leaflet of membranous domains. Immunoisolation of tuberin-bound vesicles using magnetic beads showed an enrichment of rap1, rab5, and caveolin-1, all of which have been found in specialized lipid microdomains, caveolae. Our data suggest that hamartin and tuberin are multicompartmental proteins that partially reside in caveolin-1-enriched structures and potentially affect their signaling.     

Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles

Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.     

Accumulation of rab4GTP in the cytoplasm and association with the peptidyl-prolyl isomerase pin1 during mitosis

Transport through the endocytic pathway is inhibited during mitosis. The mechanism responsible for this inhibition is not understood. Rab4 might be one of the proteins involved as it regulates transport through early endosomes, is phosphorylated by p34(cdc2) kinase, and is translocated from early endosomes to the cytoplasm during mitosis. We investigated the perturbation of the rab4 GTPase cycle during mitosis. Newly synthesized rab4 was less efficiently targeted to membranes during mitosis. By subcellular fractionation of mitotic cells, we found a large increase of cytosolic rab4 in the active GTP-form, an increase not associated with the cytosolic rabGDP chaperone GDI. Instead, phosphorylated rab4 is in a complex with the peptidyl-prolyl isomerase Pin1 during mitosis, but not during interphase. Our results show that less efficient recruitment of rab4 to membranes and a bypass of the normal GDI-mediated retrieval of rab4GDP from early endosomes reduce the amount of rab4GTP on membranes during mitosis. We propose that phosphorylation of rab4 inhibits both the recruitment of rab4 effector proteins to early endosomes and the docking of rab4-containing transport vesicles. This mechanism might contribute to the inhibition of endocytic membrane transport during mitosis.     

Rab3a is involved in transport of synaptic vesicles to the active zone in mouse brain nerve terminals

The rab family of GTP-binding proteins regulates membrane transport between intracellular compartments. The major rab protein in brain, rab3A, associates with synaptic vesicles. However, rab3A was shown to regulate the fusion probability of synaptic vesicles, rather than their transport and docking. We tested whether rab3A has a transport function by analyzing synaptic vesicle distribution and exocytosis in rab3A null-mutant mice. Rab3A deletion did not affect the number of vesicles and their distribution in resting nerve terminals. The secretion response upon a single depolarization was also unaffected. In normal mice, a depolarization pulse in the presence of Ca(2+) induces an accumulation of vesicles close to and docked at the active zone (recruitment). Rab3A deletion completely abolished this activity-dependent recruitment, without affecting the total number of vesicles. Concomitantly, the secretion response in the rab3A-deficient terminals recovered slowly and incompletely after exhaustive stimulation, and the replenishment of docked vesicles after exhaustive stimulation was also impaired in the absence of rab3A. These data indicate that rab3A has a function upstream of vesicle fusion in the activity-dependent transport of synaptic vesicles to and their docking at the active zone.     

Dissociation of complexes between 70 kDa stress proteins and presecretory proteins is facilitated by a cytosolic factor.

Members of the 70 kDa stress protein family were shown previously to facilitate the posttranslational translocation of presecretory proteins into the endoplasmic reticulum and protein precursors into mitochondria. To identify proteins that interact with 70 kDa stress proteins during the early steps of posttranslational translocation, polyclonal antibodies were raised against purified yeast cytosolic stress proteins. They were used to immunoprecipitate complexes between 70 kDa stress proteins and a radiolabeled presecretory protein, prepro-alpha-factor, that was translated in vitro. Complexes between prepro-alpha-factor and 70 kDa stress proteins were stable, but could be dissociated in the presence of ATP and crude cytosolic extracts from yeast. These results are consistent with the idea that 70 kDa stress proteins act as molecular chaperones in translocation by binding to precursor proteins before or during their passage across membranes.     

Association of Rab3A with synaptic vesicles at late stages of the secretory pathway

Rab3A is a small GTP-binding protein highly concentrated on synaptic vesicles. Like other small GTP-binding proteins it is thought to cycle between a soluble and a membrane-associated state. To determine at which stage of the life cycle of synaptic vesicles rab3A is associated with their membranes, the localization of the protein in neurons and neuroendocrine cells at different developmental and functional stages was investigated. In all cases, rab3A was colocalized with synaptic vesicle markers at the cell periphery, but was absent from the Golgi area, suggesting that rab3A associates with vesicles distally to the Golgi complex and dissociates from vesicle membranes before they recycle to this region. Immunofluorescence experiments carried out on frog motor end plates demonstrated that massive exocytosis of synaptic vesicles is accompanied by a translocation of rab3A to the cell surface. The selective localization of rab3A on synaptic vesicles at stages preceding their fusion with the plasmalemma suggests that the protein is part of a regulatory machinery that is assembled onto the vesicles in preparation for exocytosis.     

Regulation of membrane transport through the endocytic pathway by rabGTPases.

Small GTP binding proteins of the rab family are associated with the cytoplasmic surface of compartments of the central vacuolar system. Several of them, including rab5, rab4 and rab11, are localized to early endocytic organelles where they regulate distinct events in the transferrin receptor pathway. Whereas rab5 is controlling transport to early endosomes, rab4 and rab11 are involved in the regulation of recycling back to the plasma membrane. How GTP-hydrolysis of rab bound GTP is related to the role of these proteins in endocytosis is not yet known, but quick progress is being made towards this goal through the identification of proteins regulating the activity of these rab proteins.     

rab GTP-binding proteins implicated in vesicular transport are isoprenylated in vitro at cysteines within a novel carboxyl-terminal motif.

Low molecular mass GTP-binding proteins encoded by the mammalian rab genes are found in membranes of the Golgi complex and endosomes, suggesting that they play a role in the movement of exocytic and endocytic vesicles. The basis for the membrane association of these proteins has not been defined. Herein, we demonstrate that terminal cysteine residues in the rab1B, rab2, and rab5 proteins undergo thioether modification by isoprenyl groups when these proteins are translated in vitro in the presence of a radiolabeled isoprenoid precursor, [3H]mevalonate. Results of gel permeation chromatography of the radiolabeled hydrocarbons suggest that these proteins are modified specifically by isoprenyl groups of the 20-carbon diterpene class, rather than the 15-carbon farnesyl class known to be involved in modification of ras proteins. The rab1 and rab2 proteins lack the carboxyl-terminal amino acid motif common to all previously identified isoprenylated proteins, i.e. CXXX, where X is an unspecified amino acid. Analysis of altered translation products generated by site-directed mutagenesis indicates that modification of rab1B protein requires an intact carboxyl-terminal sequence consisting of GGCC. This represents a new amino acid motif for isoprenylation.     

The monomeric GTP binding protein,rab3a,is associated with the acrosome in mouse sperm

Exocytosis of the sperm acrosome is an obligate precursor to successful egg penetration and subsequent fertilization. In most mammals, acrosomal exocytosis occurs at a precise time, after sperm binding to the zona pellucida of the egg, and is induced by a specific component of the zona pellucida. It may be considered an example of regulated secretion with the acrosome of the sperm analogous to a single secretory vesicle. Monomeric G proteins of the rab3 subfamily, specifically rab3a, have been shown to be important regulators of exocytosis in secretory cells, and we hypothesized that these proteins may regulate acrosomal exocytosis. Using α[³²P] GTP binding to Immobilon blotted mouse sperm proteins, the presence of three or more monomeric GTP binding proteins was identified with M_r = 22, 24, and 26 × 10³. Alpha[³²P] GTP binding could be competed by GTP and GDP, but not GMP, ATP, or ADP. Anti‐peptide antibodies specific for rab3a were used to identify the 24 kDa G protein as rab3a. Using immunocytochemistry, rab3a was localized to the head of acrosome‐intact sperm and was lost during acrosomal exocytosis. It was identified in membrane and cytosolic fractions of sperm with the predominant form being membrane‐bound, and its membrane association did not change upon capacitation. Immunogold labeling and electron microscopy demonstrated a subcellular localization in clusters to the periacrosomal membranes and cytoplasm. These data identify the presence of rab3a in acrosomal membranes of mouse sperm and suggest that rab3a plays a role in the regulation of zona pellucida ‐induced acrosomal exocytosis. Mol. Reprod. Dev. 53:413–421, 1999. © 1999 Wiley‐Liss, Inc.     

A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion

The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(-)) had a greater affinity in vitro for rab5-guanosine 5'-O-2-(thio)diphosphate than for rab5-guanosine 5'-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(-) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5(G78A)) but not with GFP-rab5(Q79L), a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(+)) was completely associated with the particulate fraction and localized extensively with GFP-rab5(wt) in punctate endosome-like structures. Overexpression of rab5ipTM(+) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(-) inhibited rab5(wt)-stimulated endosome fusion but did not inhibit fusion stimulated by rab5(Q79L). rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5.     

Kif5B and Kifc1 interact and are required for motility and fission of early endocytic vesicles in mouse liver

Early endocytic vesicles loaded with Texas Red asialoorosomucoid were prepared from mouse liver. These vesicles bound to microtubules in vitro, and upon ATP addition, they moved bidirectionally, frequently undergoing fission into two daughter vesicles. There was no effect of vanadate (inhibitor of dynein) on motility, whereas 5'-adenylylimido-diphosphate (kinesin inhibitor) was highly inhibitory. Studies with specific antibodies confirmed that dynein was not associated with these vesicles and that Kif5B and the minus-end kinesin Kifc1 mediated their plus- and minus-end motility, respectively. More than 90% of vesicles associated with Kifc1 also contained Kif5B, and inhibition of Kifc1 with antibody resulted in enhancement of plus-end-directed motility. There was reduced vesicle fission when either Kifc1 or Kif5B activity was inhibited by antibody, indicating that the opposing forces resulting from activity of both motors are required for fission to occur. Immunoprecipitation of native Kif5B by FLAG antibody after expression of FLAG-Kifc1 in 293T cells indicates that these two motors can interact with each other. Whether they interact directly or through a complex of potential regulatory proteins will need to be clarified in future studies. However, the present study shows that coordinated activity of these kinesins is essential for motility and processing of early endocytic vesicles.     

Regulated Exocytosis in the Pancreatic Acinar Cell

The studies reported here will summarize the major events taking place during the synthesis, intracellular transport and discharge of secretory proteins from the pancreatic acinar cell. We will summarize the work that led to the definition of the regulated secretory pathway in the acinar cell followed by an update of the major steps in the pathway to incorporate new information on vesicular transport that has been gathered over the past 10 years from a number of laboratories. These studies arise from an amazing convergence of information derived from studies on the simpler eukaryote, S. cerevisiae, from biochemical analysis of neurotransmitter release, and from in vitro membrane fusion systems that have allowed for the dissection of the proteins involved in membrane recognition and fusion. Taken together, these studies have shown that the major proteins involved in membrane targeting and fusion, and the accessory proteins that control these events, are highly conserved over vast periods of evolutionary time. Thus, information derived from each of these systems and approaches can be transferred directly to regulated exocytosis in the pancreatic acinar cell — a system that has superimposed on it the complexities of organization into a polarized epithelium and control from the extracellular milieu via neurohormones. The ensuing hypothesis that integrates this body of information is termed the SNARE hypothesis. According to this hypothesis, the core complex of NSF (N-ethylmaleimide sensitive fusion protein) and SNAPs (soluble NSF attachment proteins) pair with their cognate receptors, SNAREs, present on the vesicles (v-SNARE) and the target membrane (t-SNARE) to form a complex that can lead to specific docking and fusion of the vesicles with their target membranes. This process is believed to be controlled by a variety of accessory proteins including synaptotagmin, a Ca²⁺ binding clamp for exocytosis and members of the rab family of low molecular weight GTP-binding proteins. Several of these proteins have been found by us to be present in the pancreatic acinar cell and are likely involved in similar processes that have been worked out in simpler systems. For example, we have shown that rab3D is uniquely associated with the cytosolic side of zymogen granule membranes as an integral membrane protein and that peptides from the effector domain of the rab proteins are able to induce secretion from permeabilized acinar cells, suggesting a role for this process in regulated exocytosis. These types of approaches are being used to define the localizaiton and function of members of the SNARE family of proteins and of proteins that control formation of the SNARE complex with a particular emphasis on their role in hormonally-elicited secretion. In our presentations, we will also discuss the acquisition of stimulus secretion coupling during the perinatal period in the developing rat pancreas since this system provides the possibility of defining, in a system that does not require exogenous transfection, the sequential expression of factors involved in membrane targeting and fusion. For example, during secretogenesis, rab3D is initially cytosolic at a time when the machinery of exocytosis is present but not functional, and only becomes associated with zymogen granule membranes after birth when stimulus-secretion coupling is acquired.     

Recombinant SFD isoforms activate vacuolar proton pumps.

The vacuolar proton pump of clathrin-coated vesicles is composed of two general sectors, a cytosolic, ATP hydrolytic domain (V1) and an intramembranous proton channel, V0. V1 is comprised of 8-9 subunits including polypeptides of 50 and 57 kDa, termed SFD (Sub Fifty-eight-kDa Doublet). Although SFD is essential to the activation of ATPase and proton pumping activities catalyzed by holoenzyme, its constituent polypeptides have not been separated to determine their respective roles in ATPase functions. Recent molecular characterization of these subunits revealed that they are isoforms that arise through an alternative splicing mechanism (Zhou, Z., Peng, S.-B., Crider, B.P., Slaughter, C., Xie, X.S., and Stone, D.K. (1998) J. Biol. Chem. 273, 5878-5884). To determine the functional characteristics of the 57-kDa (SFDalpha)1 and 50-kDa (SFDbeta) isoforms, we expressed these proteins in Escherichia coli. We determined that purified recombinant proteins, rSFDalpha and rSFDbeta, when reassembled with SFD-depleted holoenzyme, are functionally interchangeable in restoration of ATPase and proton pumping activities. In addition, we determined that the V-pump of chromaffin granules has only the SFDalpha isoform in its native state and that rSFDalpha and rSFDbeta are equally effective in restoring ATPase and proton pumping activities to SFD-depleted enzyme. Finally, we found that SFDalpha and SFDbeta structurally interact not only with V1, but also withV0, indicating that these activator subunits may play both structural and functional roles in coupling ATP hydrolysis to proton flow.     

rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain.

rab3A is a low molecular weight (LMW) GTP-binding protein specifically expressed in brain and localized to synaptic vesicles. rab3A has been proposed to play a role in neurotransmitter release by regulating membrane flow in the nerve terminal. In an attempt to define other LMW GTP-binding proteins that may regulate neurotransmitter release, seven cDNA clones encoding new members of the rab family of LMW GTP-binding proteins were isolated from a rat brain cDNA library. The rab proteins contain the four conserved structural domains essential for GTP binding in addition to domains required for membrane localization and effector protein interactions. One protein, rab16, is closely related to members of the rab3 subfamily, whereas two others are assigned as the rat homologs of canine rab8 and rab10. Four additional clones, rab12, rab13, rab14, and rab15, revealed unique sequences and are new members of the rab family of LMW GTP-binding proteins. The patterns of expression of rab15 and rab3A closely overlap but differ from that observed for all other known LMW GTP-binding proteins. This data suggests that rab15 may act in concert with rab3A in regulating aspects of synaptic vesicle membrane flow within the nerve terminal.     

Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.

Existing data support the hypothesis that insulin triggers the exocytosis of small vesicles containing the GluT4 isoform of the glucose transporter. The data also suggest that these vesicles reform through endocytosis of GluT4. These processes resemble those described for synaptic vesicles after depolarization of nerve cells. To determine whether GluT4 vesicles are related to synaptic vesicles, rat adipocyte low density microsomes (LDM), which are rich in GluT4 vesicles, were screened for the synaptic vesicle proteins synaptotagmin, synaptophysin, SV2, p29, rab3, and VAMP (synaptobrevin) by immunoblotting. Two polypeptides that reacted with antibodies against the VAMPs were identified, one with the same apparent size as the two isoforms of VAMP in the brain (18 kDa) and one that was slightly smaller (17 kDa). These members of the VAMP family were highly enriched in GluT4 vesicles isolated by immunoadsorption and translocated from the LDM to the plasma membrane in response to insulin. With the exception of rab3, which was observed in the LDM but was not localized in the GluT4 vesicles, the other synaptic vesicle proteins were not detected. The presence of the VAMPs in both GluT4 and synaptic vesicles suggests that the genesis and/or exocytosis of these two types of vesicles involve shared processes.     

A vacuolar sorting receptor PV72 on the membrane of vesicles that accumulate precursors of seed storage proteins (PAC vesicles)

A novel vesicle, referred to as a precursor-accumulating (PAC) vesicle, mediates the transport of storage protein precursors to protein storage vacuoles in maturing pumpkin seeds. PV72, a type I integral membrane protein with three repeats of epidermal growth factor, was found on the membrane of the PAC vesicles. PV72 had an ability to bind to pro2S albumin, a storage protein precursor, in a Ca(2+)-dependent manner, via the C-terminal region of pro2S albumin, which was found to function as a vacuolar targeting signal. This implies that PV72 is a vacuolar sorting receptor of the storage protein. PV72 was specifically and transiently accumulated at the middle stage of seed maturation in association with the synthesis of storage proteins. Subcellular fractionation showed that PV72 was also accumulated in the microsomal fraction. A fusion protein consisting of GFP and the transmembrane domain and the cytosolic tail of PV72 was localized in Golgi complex. PV72 in the isolated PAC vesicles had a complex type of oligosaccharide, indicating that PV72 passed though the Golgi complex. These results suggest that PV72 is recycled between PAC vesicles and Golgi complex/post-Golgi compartments. PV72 appears to be responsible for recruiting pro2S albumin molecules from the Golgi complex to the PAC vesicles.     

GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.