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.     

SV2 and o-rab3 Remain Associated with Recycling Synaptic Vesicles

Abstract: o-rab3 is an electric ray homologue of low molecular weight GTP-binding proteins thought to be involved in targeting of secretory vesicles to sites of exocytosis. The stimulation-dependent association of o-rab3 with synaptic vesicles was compared with that of the membrane-integral synaptic vesicle protein 2 (SV2). On application of immunoelectron microscopy and the colloidal gold technique, antibodies against either protein labeled the synaptic vesicle membrane compartment. Synaptic vesicles recycled under conditions of low frequency stimulation (0.1 Hz) retained their complement of both SV2 and o-rab3. Isolation of synaptic vesicles by density-gradient centrifugation and subsequent column chromatography yielded no indication of a stimulation-dependent release of o-rab3 from synaptic vesicles. In contrast, multivesicular bodies and vacuoles occasionally observed in the nerve terminals contained SV2 but little if any o-rab3. It is concluded that o-rab3 remains associated with the synaptic vesicle membrane compartment during stimulation-induced cycles of repeated exo- and endocytosis. o-rab3 may be lost once the vesicle enters the prelysosomal pathway.     

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.     

Quantitative proteomic profiling of the promastigotes and the intracellular amastigotes of Leishmania donovani isolates identifies novel proteins having a role in Leishmania differentiation and intracellular survival

Protozoan parasites of the genus Leishmania are important human pathogens that cycle between an extracellular promastigote stage residing in the sandflies and an intracellular amastigote stage colonizing the phagolysosomal compartment of the mammalian macrophages. Here, we used the isobaric tagging method to quantify the global proteomic differences between the promastigotes and the intracellular amastigotes of three different Leishmania donovani clones derived from the THP-1 human macrophage cell line. We identified a substantial number of differentially modulated proteins involved in nutrient acquisition and energy metabolism, cell motility and cytoskeleton, transport, cell signaling and stress response. Proteins involved in vesicular trafficking and endocytosis like the rab7 GTP binding protein, GTP-binding proteins of the Ras superfamily and developmentally regulated GTP-binding protein 1 revealed enhanced expression and also a putative dynein heavy chain protein was found to be up-regulated in the amastigotes and it probably has a role in cargo transport inside the vesicles. Significantly, in the amastigotes the expression of a protein involved in glucose transport was increased eight to fifteen-fold, whereas concentrations of several proteins associated with cell motility and cytoskeleton were reduced. Thus, the quantitative proteomic analysis of L. donovani isolates sheds light on some novel proteins that may have a role in Leishmania differentiation and intracellular survival.     

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.     

TGN38/41: a molecule on the move

TGN38/41 is a heterodimeric integral membrane protein that cycles between the trans Golgi network and the cell surface. A tyrosine-containing tetrapeptide motif within its cytoplasmic tail is necessary and sufficient for determining its steady-state location in the TGN. Recent results have shown that TGN38/41 plays an essential role in the formation of exocytic vesicles at the TGN by serving as a receptor for complexes of a cytoplasmic protein known as p62, and one of four small GTP-binding proteins, including rab6. For budding to occur, this complex must bind to the cytoplasmic domain of TGN38/41. We propose here that TGN38/41 may couple the segregation of secretory proteins to the budding of exocytic vesicles at the TGN.     

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.     

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.     

Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane

Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.     

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3- like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.     

A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons.

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding proteins of the rab family. We have used hippocampal neurons in culture and analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expression was accomplished by using sequence-specific antisense oligonucleotides. While rab8 depletion resulted in the blockage of morphological maturation in 95% of the neurons, suppression of expression of another rab protein, rab3a, had no effect, and all neurons developed normal axons and dendrites. The impairment of neuronal maturation by rab8 antisense treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we observed in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating antisense-treated neurons with Bodipy-labeled ceramide, a fluorescent marker for newly formed exocytic vesicles, we observed fluorescence labeling restricted to the Golgi apparatus, whereas in control cells labeling was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowth.     

Synthetic peptides of the effector-binding domain of rab enhance secretion from digitonin-permeabilized chromaffin cells.

There is evidence that the rab class of low molecular weight GTP-binding proteins is involved in vesicular transfer from endoplasmic reticulum to Golgi and between Golgi cisternae. To determine whether similar proteins play a role in regulated exocytosis, the effects of synthetic peptides derived from low molecular weight GTP-binding proteins on catecholamine secretion from digitonin-permeabilized chromaffin cells were investigated. The synthetic peptides represent the putative effector-binding domains of the rab, ras and ral classes of low molecular weight GTP-binding proteins and correspond to ras(33-48). Two rab peptides but neither a ras nor a ral peptide enhanced Ca(2+)-dependent secretion by approximately 30%. Maximal secretion in response to Ca2+ was increased. The enhancement was not blocked by the pseudosubstrate inhibitor of protein kinase C, PKC(19-31), thus indicating that activation of protein kinase C was not responsible for the enhancement of secretion. Similarly a rab peptide but neither a ras nor a ral peptide enhanced GppNHp-induced secretion 30-70%. The peptides had little or no effect in the absence of Ca2+ or GppNHp. The data are consistent with a protein of the rab class playing a role in regulated exocytosis.     

Role of actin in regulated exocytosis and compensatory membrane retrieval: insights from an old acquaintance

This review summarizes new insights into the role of the actin cytoskeleton in exocytosis and compensatory membrane retrieval from mammalian regulated secretory cells. Data from our lab and others now indicate that the actin cytoskeleton is involved in exocytosis both as a negative regulator of membrane fusion under resting conditions and as a facilitator of movement of secretory granules to their site of fusion with the apical plasmalemma. Coating of docked secretory granules with actin filaments correlates with the dissociation of secretory-granule-associated rab3D, pointing out a novel role for rab proteins in modulating the actin cytoskeleton during regulated exocytosis. Compensatory membrane retrieval following regulated exocytosis is also critically dependent on the actin cytoskeleton both in initiating the formation of clathrin-coated retrieval vesicles and subsequent trafficking back into the cell. We propose that insertion of secretory granule membrane into the plasmalemma initiates a trigger for membrane retrieval, possibly by exposing sites where proteins involved in compensatory membrane retrieval are assembled. The results summarized in this review were derived primarily from investigations on the pancreatic acinar cell, an old friend who is providing modern wisdom not attainable in other simpler systems.     

rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.     

Carboxyl-methylation of Rab3D in the rat pancreatic acinar tumor cell line AR42J.

Rab3D is a small GTPase implicated in regulated exocytosis, and is a marker of secretory granules in exocrine cells. We have previously shown that rab3D undergoes reversible carboxyl-methylation in adult rat pancreatic acinar cells, and that carboxyl-methylation of rab3D is developmentally regulated concomitantly with the maturation of the regulated secretory apparatus in rat pancreas. We also observed that dexamethasone treatment of the rat pancreatic acinar tumor cell line, AR42J, led to a significant increase in the size of the unmethylated pool of a rab3-like protein. The current study was designed to further characterize this rab3-like protein. Here we show that AR42J cells express rab3D, and that the protein focuses on 2D gels as two spots with pI values of 4.9 and 5.0. Treatment of AR42J cells with N-acetyl-S-geranylgeranyl-l-cysteine, an inhibitor of carboxyl-methylation, led to a decrease in the basic form of rab3D and a proportional increase in the acidic form. In contrast, N-acetyl-S-farnesyl-l-cysteine, which inhibits carboxyl-methylation of farnesylated proteins, had no effect. Lovastatin, an inhibitor of geranylgeranylation, also induced an accumulation of the acidic form of rab3D. Taken together, these data indicate that rab3D can undergo reversible carboxyl-methylation in AR42J cells by a geranylgeranyl-specific methyltransferase. The 2D gel and immunoblotting analyses indicated that dexamethasone treatment of AR42J cells led to an increase in the proportion of the unmethylated form of rab3D concurrent to inducing a regulated secretory pathway, similar to the rab3D profile change in developing rat pancreas. Our data, along with previous studies done on developing rat pancreas, indicate that the tumor cell line AR42J represents a good model system for studying the regulated secretory pathway, and that carboxyl-methylation of rab3D may play a role in the acquisition of stimulus-secretion coupling.     

Spatial segregation of the regulated and constitutive secretory pathways

Recent experiments using DNA transfection have shown that secretory proteins in AtT-20 cells are sorted into two biochemically distinct secretory pathways. These two pathways differ in the temporal regulation of exocytosis. Proteins secreted by the regulated pathway are stored in dense-core granules until release is stimulated by secretagogues. In contrast, proteins secreted by the constitutive pathway are exported continuously, without storage. It is not known whether there are mechanisms to segregate regulated and constitutive secretory vesicles spatially. In this study, we examined the site of insertion of constitutive vesicles and compared it with that of regulated secretory granules. Regulated granules accumulate at tips of processes in these cells. To determine whether constitutively externalized membrane proteins are inserted into plasma membrane at the cell body or at process tips, AtT-20 cells were infected with ts-O45, a temperature-sensitive mutant of vesicular stomatitis virus in which transport of the surface glycoprotein G is conditionally blocked in the ER. After switching to the permissive temperature, insertion of G protein was detected at the cell body, not at process tips. Targeting of constitutive and regulated secretory vesicles to distinct areas of the plasma membrane appears to be mediated by microtubules. We found that while disruption of microtubules by colchicine had no effect on constitutive secretion, it completely blocked the accumulation of regulated granules at special release sites. Colchicine also affected the proper packaging of regulated secretory proteins. We conclude that regulated and constitutive secretory vesicles are targeted to different areas of the plasma membrane, most probably by differential interactions with microtubules. These results imply that regulated secretory granules may have unique membrane receptors for selective attachment to microtubules.     

Small GTP-binding proteins associated with secretory vesicles of Paramecium

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20-30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras-like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [alpha-32P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22-31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.     

Lamellar bodies of rat alveolar type 2 cells have late endosomal marker proteins on their limiting membranes

Alveolar type 2 cells are known to take up surfactant phospholipids and proteins from the alveolar space and recycle them into secretory organelles via a receptor-mediated endocytic pathway. To clarify the intracellular route(s) through which materials ingested by the cells are processed, we examined the immunocytochemical localization of late endosomal and lysosomal membrane markers, rab 7 and lamp 1 proteins, within rat alveolar type 2 cells. The limiting membranes of lamellar bodies (LBs) showed positive immunoreactivity for both proteins, whereas multivesicular bodies (MVBs) exhibited positive immunoreactivity only for lamp 1 protein on free vesicles in the MVB lumen. From these findings, it is suggested that LBs are not only secretory granules, but also constitute one of the late endosomal compartments of the cells and that MVBs of this cell type may be targeted to cell organelle(s) other than lysosomes.