Helen Plutner

Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum.

Newly synthesized proteins are believed to move from the endoplasmic reticulum (ER) to the Golgi by bulk flow, and sorting is assumed to occur exclusively in the trans-Golgi network (TGN). Using quantitative immunoelectron microscopy, we demonstrate that vesicular stomatitis virus glycoprotein (VSV-G) is sorted from resident ER proteins and concentrated 5- to 10-fold in 40-80 nm vesicles during vesicle budding from the ER. Accumulation of VSV-G in pre-Golgi vesicular carriers is the only detectable concentration step in its transport to the TGN. From these results, it is apparent that export from the ER is not exclusively mediated by bulk flow. The ER exerts an unanticipated level of control to insure selective and efficient entry of mature protein into the secretory pathway.

Rab-αGDI activity is regulated by a Hsp90 chaperone complex

The Rab‐specific αGDP‐dissociation inhibitor (αGDI) regulates the recycling of Rab GTPases. We have now identified a novel αGDI complex from synaptic membranes that contains three chaperone components: Hsp90, Hsc70 and cysteine string protein (CSP). We find that the αGDI–chaperone complex is dissociated in response to Ca2+‐induced neurotransmitter release, that chaperone complex dissociation is sensitive to the Hsp90 inhibitor geldanamycin (GA) and that GA inhibits the ability of αGDI to recycle Rab3A during neurotransmitter release. We propose that αGDI interacts with a specialized membrane‐associated Rab recycling Hsp90 chaperone system on the vesicle membrane to coordinate the Ca2+‐dependent events triggering Rab‐GTP hydrolysis with retrieval of Rab‐GDP to the cytosol.

Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway.

Synthetic peptides of the putative effector domain of members of the ras‐related rab gene family of small GTP‐binding proteins were synthesized and found to be potent inhibitors of endoplasmic reticulum (ER) to Golgi and intra‐Golgi transport in vitro. Inhibition of transport by one of the effector domain peptides was rapid (t1/2 of 30 s), and irreversible. Analysis of the temporal site of peptide inhibition indicated that a late step in transport was blocked, coincident with a Ca2(+)‐dependent prefusion step. The results provide novel biochemical evidence for the role of members of the rab gene family in vesicular transport in mammalian cells, and implicate a role for a new downstream Rab effector protein (REP) regulating vesicle fusion.

The δ subunit of AP-3 is required for efficient transport of VSV-G from the trans-Golgi network to the cell surface

Vesicular stomatitis virus glycoprotein (VSV-G) is a transmembrane protein that functions as the surface coat of enveloped viral particles. We report the surprising result that VSV-G uses the tyrosine-based di-acidic motif (-YTDIE-) found in its cytoplasmic tail to recruit adaptor protein complex 3 for export from the trans-Golgi network. The same sorting code is used to recruit coat complex II to direct efficient transport from the endoplasmic reticulum to the Golgi apparatus. These results demonstrate that a single sorting sequence can interact with sequential coat machineries to direct transport through the secretory pathway. We propose that use of this compact sorting domain reflects a need for both efficient endoplasmic reticulum export and concentration of VSV-G into specialized post-trans-Golgi network secretory-lysosome type transport containers to facilitate formation of viral coats at the cell surface.

The mammalian guanine nucleotide exchange factor mSec12 is essential for activation of the Sar1 GTPase directing endoplasmic reticulum export.

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the endoplasmic reticulum of mammalian cells. To begin to elucidate its mechanism of action, we now report the identity of the mammalian homolog to the yeast Sec12 guanine nucleotide exchange factor (18% identity) that promotes Sar1 activation. Mammalian Sec12 (mSec12) is a type II transmembrane protein with a large cytosolic domain, a fragment of which has previously been reported as the transcription factor prolactin regulatory element binding protein (PREB). mSec12 promotes efficient guanine nucleotide exchange on Sar1, but not Arf1 or Rab GTPases. mSec12 is localized to the endoplasmic reticulum and an antibody to the cytosolic domain of mSec12 potently inhibits Sar1 recruitment and the formation of COPII vesicles in vitro. The dominant negative GDP‐restricted mutant Sar1[T39N] is shown to be a potent inhibitor of mSec12 activity, consistent with its role in preventing COPII vesicle formation in vitro and during transient expression in vivo. We propose that mSec12 is an evolutionarily distant guanine nucleotide exchange factor directing Sar1 GTPase activation in mammalian cells. Its divergence from yeast Sec12p may reflect the specialized needs of the mammalian endoplasmic reticulum involving the formation of Sar1‐dependent transitional elements (Aridor M, et al. J Cell Biol 2001;152:213–229) and selection of cargo into prebudding complexes.

The Lysophospholipid Acyltransferase Antagonist CI-976 Inhibits a Late Step in COPII Vesicle Budding

The mechanism of coat protein (COP)II vesicle fission from the endoplasmic reticulum (ER) remains unclear. Lysophospholipid acyltransferases (LPATs) catalyze the conversion of various lysophospholipids to phospholipids, a process that can promote spontaneous changes in membrane curvature. Here, we show that 2,2‐methyl‐N‐(2,4,6,‐trimethoxyphenyl)dodecanamide (CI‐976), a potent LPAT inhibitor, reversibly inhibited export from the ER in vivo and the formation of COPII vesicles in vitro. Moreover, CI‐976 caused the rapid and reversible accumulation of cargo at ER exit sites (ERESs) containing the COPII coat components Sec23/24 and Sec13/31 and a marked enhancement of Sar1p‐mediated tubule formation from ERESs, suggesting that CI‐976 inhibits the fission of assembled COPII budding elements. These results identify a small molecule inhibitor of a very late step in COPII vesicle formation, consistent with fission inhibition, and demonstrate that this step is likely facilitated by an ER‐associated LPAT.

The role of ARF1 and rab GTPases in polarization of the Golgi stack.

The organization and sorting of proteins within the Golgi stack to establish and maintain its cis to trans polarization remains an enigma. The function of Golgi compartments involves coat assemblages that facilitate vesicle traffic, Rab‐tether‐SNAP receptor (SNARE) machineries that dictate membrane identity, as well as matrix components that maintain structure. We have investigated how the Golgi complex achieves compartmentalization in response to a key component of the coat complex I (COPI) coat assembly pathway, the ARF1 GTPase, in relationship to GTPases‐regulating endoplasmic reticulum (ER) exit (Sar1) and targeting fusion (Rab1). Following collapse of the Golgi into the ER in response to inhibition of activation of ARF1 by Brefeldin A, we found that Sar1‐ and Rab1‐dependent Golgi reformation took place at multiple peripheral and perinuclear ER exit sites. These rapidly converged into immature Golgi that appeared as onion‐like structures composed of multiple concentrically arrayed cisternae of mixed enzyme composition. During clustering to the perinuclear region, Golgi enzymes were sorted to achieve the degree of polarization within the stack found in mature Golgi. Surprisingly, we found that sorting of Golgi enzymes into their subcompartments was insensitive to the dominant negative GTP‐restricted ARF1 mutant, a potent inhibitor of COPI coat disassembly and vesicular traffic. We suggest that a COPI‐independent, Rab‐dependent mechanism is involved in the rapid reorganization of resident enzymes within the Golgi stack following synchronized release from the ER, suggesting an important role for Rab hubs in directing Golgi polarization.

Transport of protein between endoplasmic reticulum and Golgi compartments in semiintact cells

Publisher Summary This chapter discusses the transport of protein between endoplasmic reticulum and golgi compartments in semiintact cells. Preparation of cells that actively transport VSV G protein are most thoroughly characterized using CHO cells and CHO cytosol. However, there is considerable variability in transport using different cell lines or conditions. Where indicated in the procedures, measurement of transport using different marker proteins, or the use of different cell lines or cytosol preparations, may need to be optimized to obtain maximal efficiencies of transport. Extensive fragmentation and lysis of the ER (particularly in the case of strongly adherent cells) generally result in reduced transport. In particular, perforation conditions can lead to the release of soluble marker proteins from the ER during preparation of semiintact cells. In contrast, poor perforation leads to a transport that is efficient, but cytosol independent. In the latter case, cytosol dependence can be enhanced by gently homogenizing the semiintact cells with a loose-fitting pestle in a glass Dounce (10-20 strokes) prior to washing.

Preparation of semiintact cells for study of vesicular trafficking in vitro

Publisher Summary This chapter focuses on techniques used in the laboratory to reconstitute endoplasmic reticulum (ER)-to-Golgi transport in vitro, using adherent tissue culture cells that have been rendered semiintact by gently scraping them from the dish after hypotonic swelling. This technique is discussed in the context of other protocols that allow the investigator to perforate both adherent and nonadherent cells. The ER-to-Golgi transport assay is based on the observation that individual N-linked oligosaccharide-processing enzymes reside in distinct subcellular compartments.Potential markers include endogenous proteins, other viral glycoproteins, and proteins acquired through transfection. Moreover, because both the interior and exterior membranes of the cell are jointly accessible to a wide range of reagents and macromolecules, semiintact cells may provide a useful model system for the study of a broad range of problems in cell biology, including signal transduction, organization of the cytoskeletal matrix, and gene activation.

Microsome-Based Assay for Analysis of Endoplasmic Reticulum to Golgi Transport in Mammalian Cells

Publisher Summary The trafficking of proteins along the first stage of the secretory pathway is mediated by small vesicles that bud from the endoplasmic reticulum (ER) and subsequently fuse with the cis-Golgi compartment. To follow vesicle formation, a differential centrifugation procedure is employed to separate the more rapidly sedimenting ER and Golgi membranes from the slowly sedimenting vesicles. Consumption is analyzed using a two-stage assay in which vesicles isolated by differential centrifugation during stage 1 are subsequently added to stage 2 (fusion) reactions containing acceptor Golgi membranes. Use a pipette to transfer the cells to 50-ml plastic tubes and then repeat the scraping procedure to ensure that all the cells are collected. Centrifuge at 720xg for 3 min and remove the supernatant by aspiration. Resuspend each cell pellet in 0.9 ml of homogenization buffer supplemented with PIC and homogenize by three complete passes through a 1-ml ball-bearing homogenizer. Remove the supernatants by aspiration, wash the pellets with 2 ml of transport buffer, and combine the membranes into two 1.5-ml microfuge tubes.