Vesicular Budding Research Articles

Confocal imaging and tracking of the exocytotic routes for D‐serine‐mediated gliotransmission

D-Serine is an astrocyte-derived regulator for N-methyl-D-aspartate receptors, but the intracellular routes of its trafficking are still largely unknown. Here, we combined confocal microscopy with colocalization quantification to track the astrocytic organelles that store D-serine. We report that D-serine colocalizes with the transfected eGFP-synaptobrevin/VAMP2 and eGFP-cellubrevin/VAMP3, two v-SNAREs of the regulated secretory pathway. No significant colocalization was found with markers of the endosomal sorting and recycling system: EEA1, eGFP-endobrevin/VAMP8, eGFP-TI-VAMP/VAMP7, LAMP1, and CD63. Blockade of vesicular budding with colchicine shows that secretory vesicles import D-serine downstream to the Golgi apparatus. Finally, treatment of astrocytes with the Ca2+-ionophore A23187, glutamate agonists, or bradykinin trigger translocation of synaptobrevin/VAMP2 to the plasma membrane with a concomitant disappearance of D-serine from the regulated secretory pathway. Our results provide morphological evidence for a vesicular storage of D-serine in the regulated secretory pathway and the possible recruitment of these stores by Ca2+ mobilization to release D-serine.

Functional Immobilization of the Small GTPase Rab6A on DNA–Gold Nanoparticles by Using a Site‐Specifically Attached Poly(ethylene glycol) Linker and Thiol Place‐Exchange Reaction

Efficient strategies to achieve functional immobilization of biomacromolecules, such as proteins and DNA, on inorganic nanoparticles have been the subject of growing interest in recent years, since they form a basis for the rational design of functional nanomaterials. 2] These materials have a broad scope of application in bioanalytics and biomolecular electronics. However, the functional immobilization of proteins on nanoparticles is often difficult to achieve because the occurrence of multiple functional groups in proteins complicates the task of linking them to surfaces while keeping them fully functional. Commonly employed methods often take advantage of nonspecific absorption or covalent attachment. These strategies often lead to uncontrolled immobilization and random orientation of the proteins on the nanoparticle surface. One strategy to overcome the potential problems arising from these approaches is based on specific interactions between the protein and a functionalized nanoparticle surface. We report here on the controlled assembly of covalently linked protein–nanoparticle conjugates through an approach based on the highly specific modification of proteins with a functionalized poly(ethylene glycol) linker molecule by a native chemical ligation reaction (Scheme 1). Such PEGbased linkers are chemically inert and water soluble; this renders them highly attractive as solvent-exposed spacers that can easily react with nanoparticle surfaces through an appended thiol group. In this study, we investigated this strategy by using a small GTPase of the Rab family. Rab proteins are central regulators of vesicular transport and are involved in vesicular budding, targeting, and fusion. During their normal mode of action, these Rab proteins cycle between an active GTPand an inactive GDP-bound conformation; this imposes temporal and spatial regulation on membrane transport. Rab6 was originally identified as a key player of microtubule-dependent retrograde traffic, controlling vesicular trafficking from early endosomes to the trans-Golgi network, through the Golgi apparatus, and from the Golgi to the endoplasmatic reticulum,

Gastrointestinal Tumors: Metastasis and Tetraspanins

Tumors of the gastrointestinal tract -- gastric, colorectal, pancreatic and liver tumors -- account for over 50 % of cancer worldwide. The 5-year survival rate varies from > 50 % in colorectal to < 1 % in pancreatic cancer. The high cancer death rate strikingly correlates with the high metastasizing capacity of most gastrointestinal tumors. Therefore and because during the last decade several important hypotheses on metastasis formation could be settled on solid experimental ground, this review will first provide a brief outline on the currently most accepted view of tumor progression and then discuss whether and how the rather new family of tetraspanin molecules might contribute to cancer progression. Notably, some members of this family, in particular, CD82/KAI1 are known as metastasis suppressor genes, while others like CD151 and CO-029 are supposed to promote metastasis formation. The underlying mechanisms are beginning to become unraveled. Tetraspanins assemble complexes of different tetraspanins, integrins and additional transmembrane molecules in microdomains that serve as signaling platform. By creating proximity, tetraspanins modulate functional activity of the associating molecules. In addition, tetraspanins actively contribute to the intracellular traffic of the associating molecules that includes vesicular budding and formation of exosomes that are particularly rich in tetraspanins. Accordingly, the association of certain tetraspanins with the metastatic phenotype as well as the definition of other tetraspanins as metastasis suppressor genes has to be viewed from the perspective of molecular complexes rather than the individual tetraspanin.

Presenilin-1 uses phospholipase D1 as a negative regulator of β-amyloid formation

Presenilin (PS1/PS2) is a major component of gamma-secretase, the activity that mediates proteolysis of beta-amyloid precursor protein to generate beta-amyloid (Abeta). Here we demonstrate that PS1, through its loop region, binds to phospholipase D1 (PLD1), thereby recruiting it to the Golgi/trans-Golgi network. Overexpression of wild-type PLD1 reduces Abeta generation. Conversely, down-regulation of endogenous PLD1 by small hairpin RNA elevates Abeta production. The Abeta-lowering effect of PLD1 is independent of its ability to promote vesicular budding of beta-amyloid precursor protein. The data indicate that overexpression of PLD1 decreases, and down-regulation of PLD1 increases, the catalytic activity, and the association of the subunits, of gamma-secretase.

Rab7 Activity Affects Epidermal Growth Factor:Epidermal Growth Factor Receptor Degradation by Regulating Endocytic Trafficking from the Late Endosome

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family. Ligand (epidermal growth factor or EGF) binding to the EGFR results in the coordinated activation and integration of biochemical signaling events to mediate cell growth, migration, and differentiation. One mechanism the cell utilizes to orchestrate these events is ligand-mediated endocytosis through the canonical clathrin-mediated endocytic pathway. Identification of proteins that regulate the intracellular movement of the EGF.EGFR complex is an important first step in dissecting how specificity of EGFR signaling is conferred. We examined the role of the small molecular weight guanine nucleotide-binding protein (G-protein) rab7 as a regulator of the distal stages of the endocytic pathway. Through the transient expression of activating and inactivating mutants of rab7 in HeLa cells, we have determined that rab7 activity directly correlates with the rate of radiolabeled EGF and EGFR degradation. Furthermore, when inhibitory mutants of rab7 are expressed, the internalized EGF.EGFR complex accumulates in high-density endosomes that are characteristic of the late endocytic pathway. Thus, we conclude that rab7 regulates the endocytic trafficking of the EGF.EGFR complex by regulating its lysosomal degradation.

In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding.

The matrix protein VP40 plays a critical role in Ebola virus assembly and budding, a process that utilizes specialized membrane domains known as lipid rafts. Previous studies with purified protein suggest a role for oligomerization of VP40 in this process. Here, we demonstrate VP40 oligomers in lipid rafts of mammalian cells, virus-like particles, and in the authentic Ebola virus. By mutagenesis, we identify several critical C-terminal sequences that regulate oligomerization at the plasma membrane, association with detergent-resistant membranes, and vesicular release of VP40, directly linking these phenomena. Furthermore, we demonstrate the active recruitment of TSG101 into lipid rafts by VP40. We also report the successful application of the biarsenic fluorophore, FlAsH, combined with a tetracysteine tag for imaging of Ebola VP40 in live cells.

Sterols block binding of COPII proteins to SCAP, thereby controlling SCAP sorting in ER.

Sterols inhibit their own synthesis in mammalian cells by blocking the vesicular endoplasmic reticulum-to-Golgi transport of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), a sterol-sensing protein that escorts SREBPs. Unable to reach the Golgi, SREBPs are not processed by Golgi-resident proteases, and they fail to activate genes required for cholesterol synthesis. The current studies were designed to reveal whether sterols block SCAP movement by inhibiting synthesis of special vesicles dedicated to SCAP, or whether sterols block SCAP incorporation into common coat protein (COP)II-coated vesicles. Through immunoisolation, we show that SCAP-containing vesicles, formed in vitro, also contain vesicular stomatitis virus glycoprotein (VSVG) protein, a classic marker of COPII-coated vesicles. Sterols selectively block incorporation of SCAP into these vesicles without blocking incorporation of VSVG protein. We show that the mammalian vesicular budding reaction can be reconstituted by recombinant yeast COPII proteins that support incorporation of SCAP as well as VSVG into vesicles. Sterols block SCAP incorporation into vesicles by blocking Sar1-dependent binding of the COPII proteins Sec 23/24 to SCAP. These studies demonstrate feedback control of a biosynthetic pathway by the regulated binding of COPII proteins to an endoplasmic reticulum-to-Golgi transport protein.

Resting (Basal) Secretion of Proteins Is Provided by the Minor Regulated and Constitutive-like Pathways and Not Granule Exocytosis in Parotid Acinar Cells

Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as an in vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or beta-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types.

Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and Abc1-deficient mice.

Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.

Fine structure and plasticity of barosensitive neurons in the nucleus of solitary tract

Intravenous phenylephrine (PE) activates neurons in the nucleus of the solitary tract (NTS) whose distribution conforms to those of central projections of the carotid sinus and aortic depressor nerves. This was exploited to permit fine structural characterization of cells presumed to compose the first station in the processing of arterial baroreceptor input, and their responses to stimulation. Rats were perfused at varying intervals after PE injection, and sections through the baroreceptor afferent zone of the NTS prepared for preembedding immunolocalization of Fos-immunoreactivity. Labeled neurons composed a continuous strip extending from the dorsal part of the commissural NTS (NTScom) to the dorsal subnucleus at the level of the area postrema (NTSap). PE-sensitive neurons in these regions were medium-sized, round to ovoid in shape, with scant cytoplasm and an unremarkable complement of organelles. Distinctive features included extensively invaginated nuclei and well-developed Golgi apparati; Fos-ir cells in the NTSap were distinguished from those in NTScom by virtue of better-developed rough endoplasmic reticulum and Golgi, and less convoluted nuclei. Proximal synaptic input to PE-sensitive neurons was sparse and was provided by terminals containing predominantly small, clear synaptic vesicles that formed mainly symmetric junctions with somata and primary dendrites. Prolonged stimulation was accompanied by accentuation of nuclear invaginations, marked accumulation of heterochromatin at their apices, and evidence of enhanced Golgi activity (vesicular budding). These may represent adaptations to facilitate changes in gene expression, to maintain neurotransmitter availability, or both, in the face of a persistent hypertensive challenge.

Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat

The regulation of intracellular vesicular trafficking is mediated by specific families of proteins that are involved in vesicular budding, translocation, and fusion with target membranes. We purified a vesicle-associated protein from hepatic microsomes using sequential column chromatography and partially sequenced it. Oliogonucleotides based on these sequences were used to clone the protein from a rat liver cDNA library. The clone encoded a novel protein with a predicted mass of 137 kDa (p137). The protein had an N terminus WD repeat motif with significant homology to Sec31p, a member of the yeast COPII coat that complexes with Sec13p. We found that p137 interacted with mammalian Sec13p using several approaches: co-elution through sequential column chromatography, co-immunoprecipitation from intact cells, and yeast two-hybrid analysis. Morphologically, the p137 protein was localized to small punctate structures in the cytoplasm of multiple cultured cell lines. When Sec13p was transfected into these cells, it demonstrated considerable overlap with p137. This overlap was maintained through several pharmacological manipulations. The p137 compartment also demonstrated partial overlap with ts045-VSVG protein when infected cells were incubated at 15 degrees C. These findings suggest that p137 is the mammalian orthologue of Sec31p.

The Golgi complex: perspectives and prospectives

We now have considerable understanding of the role of the Golgi complex in the posttranslational modifications of membrane and secretory proteins and of lysosomal hydrolases. It is now also clear that the Golgi plays a key role in the intracellular packaging, addressing, and sorting of these classes of proteins to their final destinations on the secretory and endocytic pathways. While it has been proposed that vesicular budding and fusion underlie entry of proteins into the Golgi from the ER and subsequent movement among its cisternae and exit to their final stations, recent observations indicate that this model may need to be revised based on studies in living cells where vesicular-tubular structures appear to mediate membrane trafficking. This will be a major challenge for investigators in the coming years who will rely again on the use of morphologic techniques of the sort that started it all in 1898.

A novel SNARE complex implicated in vesicle fusion with the endoplasmic reticulum.

Intracellular vesicular traffic is controlled in part by v- and t-SNAREs, integral membrane proteins which allow specific interaction and fusion between vesicles (v-SNAREs) and their target membranes (t-SNAREs). In yeast, retrograde transport from the Golgi complex to the ER is mediated by the ER t-SNARE Ufe1p, and also requires two other ER proteins, Sec20p and Tip20p, which bind each other. Although Sec20p is not a typical SNARE, we show that both it and Tip20p can be co-precipitated with Ufe1p, and that a growth-inhibiting mutation in Ufe1p can be compensated by a mutation in Sec20p. Furthermore, Sec22p, a v-SNARE implicated in forward transport from ER to Golgi, co-precipitates with Ufe1p and Sec20p, and SEC22 acts as an allele-specific multicopy suppressor of a temperature-sensitive ufe1 mutation. These results define a new functional SNARE complex, with features distinct from the plasma membrane and cis-Golgi complexes previously identified. They also show that a single v-SNARE can be involved in both anterograde and retrograde transport, which suggests that the mere presence of a particular v-SNARE may not be sufficient to determine the preferred target for a transport vesicle.

Plasma membrane targetting, vesicular budding and release of galectin 3 from the cytoplasm of mammalian cells during secretion.

Galectin 3, a 30 kDa galactoside-binding protein distributed widely in epithelial and immune cells, contains no signal sequence and is externalized by a mechanism independent of the endoplasmic reticulum (ER)-Golgi complex. We show here that hamster galectin 3 overexpressed in transfected cos-7 cells is secreted at a very low rate. A chimaera of galectin 3 fused to the N-terminal acylation sequence of protein tyrosine kinase p56(lck), Nt-p56(lck)-galectin 3, which is myristoylated and palmitoylated and rapidly transported to plasma membrane domains, is efficiently released from transfected cells indicating that movement of cytoplasmic galectin 3 to plasma membrane domains is a rate limiting step in lectin secretion. N-terminal acylation is not sufficient for protein secretion since p56(lck) and the chimaera Nt-p56(lck)-CAT are not secreted from transfected cells. The amino-terminal half of galectin 3 is sufficient to direct export of a chimaeric CAT protein indicating that part of the signal for plasma membrane translocation lies in the N-terminal domains of the lectin. Immunofluorescence studies show that Nt-p56(lck)-galectin 3 aggregates underneath the plasma membrane and is released by membrane blebbing. Vesicles of low buoyant density isolated from conditioned medium are enriched in galectin 3. The lectin is initially protected from exogenous collagenase but is later released in soluble protease-sensitive form from the lectin-loaded vesicles. Using murine macrophages, which secrete their endogenous galectin 3 at a moderate rate especially in the presence of Ca2+-ionophores, we were also able to trap a galectin 3-loaded vesicular fraction which was released into the culture supernatant.

Regulated Exocytosis in Chromaffin Cells: A POTENTIAL ROLE FOR A SECRETORY GRANULE-ASSOCIATED ARF6 PROTEIN

The ADP-ribosylation factor (ARF) GTP-binding proteins are believed to function as regulators of vesicular budding and fusion along the secretory pathway. To investigate the role of ARF in regulated exocytosis, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation and immunoreplica analysis. We found that ARF6 is specifically associated with the membrane of purified secretory chromaffin granules. Chemical cross-linking and immunoprecipitation experiments suggested that ARF6 may be part of a complex with betagamma subunits of trimeric G proteins. Stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid dissociation of ARF6 from secretory granules. This effect could be inhibited by AlF4- which selectively activates trimeric G proteins. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 strongly inhibited calcium-evoked secretion in streptolysin-O-permeabilized chromaffin cells. The possibility that ARF6 plays a role in the effector pathway by which trimeric G proteins control exocytosis in chromaffin cells is discussed.

2H-NMR and DSC study of DPPC-DODAB mixtures

The phase behaviour of vesicles consisting of binary mixtures of 1,2-dipalmitoyl-d 62- sn-glycero-3-phosphocholine (DPPC-d 62) and dioctadecyl-dimethyl-ammonium-bromide (DODAB) has been investigated using deuterium nuclear magnetic resonance ( 2H-NMR), differential scanning calorimetry (DSC) and freeze-fracture electron microscopy. Using both multilamellar vesicle (MLV) and solid supported vesicle (SSV) samples in our study we propose partial phase diagrams for each model system and gain information about the effect of the solid state surface (silica) on the mixing behavior. DODAB, being a cationic lipid-like amphiphile, exhibits a peculiar phase diagram when mixed with DPPC. An equimolar mixture shows a higher melting temperature than each of the pure components together with a narrow coexistence region. All other mixing ratios studied exhibit significantly broader coexistence regions and lower transition temperatures. For the MLV samples freeze fracture electron microscopy shows a reversible reduction in vesicular size by an order of magnitude in going from the fluid to the gel state. For the SSV, we observed a markedly different phase behavior compared to that of the MLV that can be ascribed to the contribution of the Coulomb interaction between bilayer and solid support to the mixing behavior, possibly leading to an asymmetry in bilayer composition. As for the MLV, the equimolar SSV sample shows a remarkable phase transition at high temperature connected with the reversible formation of highly curved structures like vesicular buds or tethers on the SSV surface.

The Effects of Brefeldin A on the Glucose Transport System in Rat Adipocytes: IMPLICATIONS REGARDING THE INTRACELLULAR LOCUS Of INSULIN-SENSITIVE Glut4

Insulin activates glucose transport by recruiting Glut4 glucose transporters from an intracellular pool to plasma membrane (PM). To localize intracellular translocating Glut4, we studied the effects of brefeldin A (BFA), which disassembles Golgi and prevents trans-Golgi vesicular budding, on the glucose transport system. Isolated rat adipocytes were treated with and without both BFA (10 micrograms/ml) and insulin. BFA did not affect maximal rates of either 2-deoxyglucose or 3-O-methyl-glucose transport or the insulin:glucose transport dose-response curve but did increase basal transport by approximately 2-fold (p < 0.05). We also measured Glut4 in PM, low (LDM) and high density microsome subfractions. In basal cells, BFA increased PM Glut4 by 58% concomitant with a 18% decrease in LDM (p < 0.05). Insulin alone increased PM Glut4 by 3-fold concomitant with a 56% decrease in LDM. BFA did not affect insulin-induced changes in Glut4 levels in PM or LDM. Most intracellular Glut4 was localized to sub-PM vesicles by immunoelectron microscopy in basal cells, and BFA did not affect insulin-mediated recruitment of immunogold-labeled Glut4 to PM. In summary, 1) in basal cells, BFA led to a small increase in glucose transport activity and redistribution of a limited number of transporters from LDM to PM; 2) BFA did not affect insulin's ability to stimulate glucose transport or recruit normal numbers of LDM Glut4 to PM; and 3) insulin action is predominantly mediated by a BFA-insensitive pool of intracellular Glut4, which localizes to sub-PM vesicles. Thus, the major translocating pool of Glut4 in rat adipocytes does not involve trans-Golgi.

Formation of membrane domains created during the budding of vesicular stomatitis virus. A model for selective lipid and protein sorting in biological membranes.

Vesicular stomatitis virus buds from domains of the plasma membrane that have a unique protein and lipid composition. Fluorescence digital imaging microscopy and resonance energy transfer were used to determine how the two viral envelope-associated proteins, the G and the M proteins, could alter the lateral distribution of lipids in large unilamellar vesicles and form domains. The G protein formed large domains in vesicles containing phosphatidic acid but not with phosphatidylserine, while the M protein formed domains enriched in both acidic phospholipids. Domains enriched in sphingomyelin were observed only when both the G protein and the M protein were present in vesicles containing phosphatidic acid. Phosphatidylcholine and gramicidin (chosen to represent a host membrane protein) were excluded from the domains. Cholesterol was induced to partition into the domains only in vesicles containing phosphatidic acid and sphingomyelin along with both of the proteins. Phosphatidylethanolamine was not enriched or depleted in the domains. Domains of similar composition were formed using vesicles made from dioleoylphospholipids and the lipids extracted from BHK-21 plasma membranes, indicating that the fatty acid composition was not as important as the polar head groups of the phospholipids. The phospholipid and cholesterol compositions of the domains formed by the G and the M proteins in vesicles were very similar to the composition of the viral envelope, suggesting that the domains represent the areas in the plasma membrane where the virus buds. This study provides a model for selective lipid and protein sorting that occurs in biological membranes.

Immunocytochemical localization of β-COP to the ER-Golgi boundary and the TGN

Recent data strongly suggest that the coatomer (COP) complex is involved in membrane transport between the ER and Golgi complex. This vesicular coat has been implicated in ER to Golgi, in intra Golgi as well as in Golgi to ER traffic. In this study we present a detailed immunocytochemical analysis of the distribution of beta-COP in different tissue culture cells. Our results extend previous studies by showing, using electron microscopy, that beta-COP accumulates on vesicular profiles and buds in the intermediate compartment (IC) under conditions that block ER to Golgi transport (15 degrees C). Importantly, under these conditions beta-COP co-localizes on these structures with a passenger protein, the membrane glycoprotein of vesicular stomatis virus (ts-O45-G). Furthermore, quantitative immunofluorescence microscopy of cells with ts-045-G accumulated in the ER, IC and trans-Golgi network, shifted briefly to the permissive temperature, showed that beta-COP was associated with many of the putative transport intermediates containing the viral glycoprotein which is in transit between the ER/IC and the cis-Golgi. The simplest interpretation of these data is that COP-coated vesicles are involved in anterograde transport of ts-045-G from the IC to the Golgi complex. Since many putative COP vesicle lacked the G protein following release of the 15 degrees C block this pool could be involved in retrograde transport. We also show that beta-COP is present on the membranes of the trans-Golgi network. However, in contrast to the ER-Golgi boundary, we could find no convincing evidence that this pool of beta-COP is associated with buds or trans-Golgi network-derived transport vesicles.

Hsp47 and Cyclophilin B Traverse the Endoplasmic Reticulum with Procollagen into Pre-Golgi Intermediate Vesicles: A ROLE FOR Hsp47 AND CYCLOPHILIN B IN THE EXPORT OF PROCOLLAGEN FROM THE ENDOPLASMIC RETICULUM

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.