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W E Balch

Publications and source records attributed to W E Balch.

At least 55 records · Page 3Linked to original sources
PubMed1997

Mss4 does not function as an exchange factor for Rab in endoplasmic reticulum to Golgi transport.

Mss4 and its yeast homologue, Dss4, have been proposed to function as guanine nucleotide exchange factors (GEFs) for a subset of Rab proteins in the secretory pathway. We have previously shown that Rab1A mutants defective in GTP-binding potently inhibit endoplasmic reticulum to Golgi transport, presumably by sequestering an unknown GEF regulating its function. We now demonstrate that these mutants stably associate with Mss4 both in vivo and in vitro and that Mss4 effectively neutralizes the inhibitory activity of the Rab1A mutants. An equivalent Rab3A mutant (Rab3A[N135I]), a Rab protein specifically involved in regulated secretion at the cell surface, associates with Mss4 as efficiently as the Rab1A[N124I] mutant. Although Rab3A[N135I] prevents the ability of Mss4 to neutralize the inhibitory effects of Rab1A mutants on transport, it has no effect on Rab1 function or endoplasmic reticulum to Golgi transport. Furthermore, quantitative immunodepletion of Mss4 fails to inhibit transport in vitro. We conclude that Mss4 and its yeast homologue, Dss4, are not GEFs mediating activation of Rab, but rather, they interact with the transient guanine nucleotide-free state, defining a new class of Ras-superfamily GTPase effectors that function as guanine nucleotide-free chaperones (GFCs).

Biological Transport↗
PubMed1996

Rab2 is essential for the maturation of pre-Golgi intermediates.

The small GTPase Rab2 is a resident of pre-Golgi intermediates and required for protein transport from the endoplasmic reticulum (ER) to the Golgi complex (Tisdale, E. J., Bourne, J. R., Khosravi-Far, R. , Der, C. J., and Balch, W. E. (1992) J. Cell Biol. 119, 749-761). The Rab2 protein, like all small GTPases, contains conserved GTP-binding domains as well as hypervariable carboxyl-terminal and amino-terminal domains. While the role of the carboxyl terminus in specific membrane localization is well recognized, the potential role of the variable NH2 terminus remains to be clarified. To determine whether the NH2 terminus of Rab2 was required for its activity in vivo, a trans dominant mutant of Rab2 that inhibits ER to Golgi transport was progressively truncated and analyzed for its effect on vesicular stomatitis virus glycoprotein transport in a vaccinia-based transient expression system. Deletion of the first 14 amino-terminal residues resulted in the loss of the inhibitory properties of the mutant without affecting its post-translational processing or membrane association. To assess the potential role of the NH2 terminus in Rab2 function, a peptide corresponding to the first 13 amino acids following the initiator methionine was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the ER to the Golgi stack. This peptide was a potent inhibitor of transport. Biochemical and morphological studies revealed that the peptide strongly interfered with assembly of pre-Golgi intermediates which mediate segregation of anterograde and retrograde transported proteins en route to the Golgi. The combined results suggest that the NH2 terminus of Rab2 is required for its function and for direct interaction with components of the transport machinery involved in the maturation of pre-Golgi intermediates.

Amino Acid Sequence↗
PubMed1996

Mammalian Sly1 regulates syntaxin 5 function in endoplasmic reticulum to Golgi transport.

Members of the syntaxin gene family are components of protein complexes which regulate vesicle docking and/or fusion during transport of cargo through the secretory pathway of eukaryotic cells. We have previously demonstrated that syntaxin 5 is specifically required for endoplasmic reticulum to Golgi transport (Dascher, C., Matteson, J., and Balch, W. E.(1994) J. Biol. Chem. 269, 29363-29366). To extend these observations we have now cloned a protein from rat liver membranes which forms a native complex with syntaxin 5. We demonstrate that this protein is the mammalian homologue to yeast Sly1p, previously identified as a protein which genetically and biochemically interacts with the small GTPase Ypt1p and Sed5p, proteins involved in docking/fusion in the early secretory pathway of yeast. Using transient expression we find that overexpression of rat liver Sly1 (rSly1) can neutralize the dominant negative effects of excess syntaxin 5 on endoplasmic reticulum to Golgi transport. These results suggest that rSly1 functions to positively regulate syntaxin 5 function.

Amino Acid Sequence↗
PubMed1996

Structure and mutational analysis of Rab GDP-dissociation inhibitor.

The crystal structure of the bovine alpha-isoform of Rab GDP-dissociation inhibitor (GDI), which functions in vesicle-membrane transport to recycle and regulate Rab GTPases, has been determined to a resolution of 1.81 A. GDI is constructed of two main structural units, a large complex multisheet domain I and a smaller alpha-helical domain II. The structural organization of domain I is surprisingly closely related to FAD-containing monooxygenases and oxidases. Sequence-conserved regions common to GDI and the choroideraemia gene product, which delivers Rab to catalytic subunits of Rab geranylgeranyltransferase II, are clustered on one face of the molecule. The two most sequence-conserved regions, which form a compact structure at the apex of GDI, are shown by site-directed mutagenesis to play a critical role in the binding of Rab proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Principles of selective transport: coat complexes hold the key.

The transport of protein through the secretory pathway of eukaryotic cells relies on small vesicular carriers, which mediate the movement of cargo between different compartments. Here, Meir Aridor and Bill Balch summarize what is currently known about the role of the cytosolic coat complexes in directing the formation and selective composition of vesicular carriers, and propose that a selective-transport model should now form the basis for study of membrane traffic in the secretory pathway.

Journal Article↗
PubMed1996

Structural insights into the function of the Rab GDI superfamily.

The 1.81 A crystal structure of Rab GDP-dissociation inhibitor (GDI), a protein that plays a critical role in the recycling of Rab GTPases involved in membrane vesicular transport, has been recently determined. Biochemical studies implicate a highly conserved region involved in Rab binding, which is common to both GDI and the evolutionarily-related choroideremia gene product (CHM/REP) required for Rab prenylation. Here, we summarize the mechanisms by which members of the GDI superfamily might function to coordinate events leading to membrane fusion, and we discuss the unexpected, yet striking structural homology of GDI to FAD-binding proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

The organization of endoplasmic reticulum export complexes.

Export of cargo from the ER occurs through the formation of 60-70nm COPII-coated vesicular carriers. We have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of ER export sites in vivo and in vitro. We find that ER buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. The basic organization of an export complex can be divided into an active COPII-containing budding zone on a single ER cisterna, which is adjacent to budding zones found on distantly connected ER cisternae. These budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain COPI coats involved in retrograde transport. Vesicles within these export complexes contain concentrated cargo molecules. The structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. We conclude that budding from the ER through recruitment of COPII is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by COPI.

Animals↗
PubMed1996

COPII vesicles derived from mammalian endoplasmic reticulum microsomes recruit COPI.

ER to Golgi transport requires the function of two distinct vesicle coat complexes, termed COPI (coatomer) and COPII, whose assembly is regulated by the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, respectively. To address their individual roles in transport, we have developed a new assay using mammalian microsomes that reconstitute the formation of ER-derived vesicular carriers. Vesicles released from the ER were found to contain the cargo molecule vesicular stomatitis virus glycoprotein (VSV-G) and p58, an endogenous protein that continuously recycles between the ER and pre-Golgi intermediates. Cargo was efficiently sorted from resident ER proteins during vesicle formation in vitro. Export of VSV-G and p58 were found to be exclusively mediated by COPII. Subsequent movement of ER-derived carriers to the Golgi stack was blocked by a trans-dominant ARF1 mutant restricted to the GDP-bound state, which is known to prevent COPI recruitment. To establish the initial site of coatomer assembly after export from the ER, we immunoisolated the vesicular intermediates and tested their ability to recruit COPI. Vesicles bound coatomer in a physiological fashion requiring an ARF1-guanine nucleotide exchange activity. These results suggest that coat exchange is an early event preceding the targeting of ER-derived vesicles to pre-Golgi intermediates.

ADP-Ribosylation Factor 1↗
PubMed1996

AP-2-containing clathrin coats assemble on mature lysosomes.

Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment.

Animals↗
PubMed1995

Ricin cytotoxicity is sensitive to recycling between the endoplasmic reticulum and the Golgi complex.

Cytotoxic proteins that kill mammalian cells by catalytically inhibiting protein synthesis must enter the cytosol in order to reach their substrates. With the exception of diphtheria toxin, which enters the cytosol from acidified endosomes, the intracellular site of translocation of other toxins including ricin, Escherichia coli Shiga-like toxin-1, and Pseudomonas exotoxin A is likely to involve early compartments of the secretory pathway. We have used a molecular approach to identify the site and mechanism of toxin delivery to the cytosol by transiently expressing mutant GTPases that inhibit the assembly of biochemical complexes mediating anterograde and retrograde transport in the exocytic and endocytic pathways. The results provide evidence to suggest that receptors actively recycling between the endoplasmic reticulum and terminal Golgi compartments are essential for toxin translocation to the cytosol from the endoplasmic reticulum. The rapid kinetics of intoxication demonstrate a substantial level of bidirectional membrane flow and sorting through the early secretory pathway.

Amino Acid Sequence↗
PubMed1995

Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells.

Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene, in the targeting of newly synthesized proteins to the vacuole, an organelle functionally equivalent to mammalian lysosomes (Schu, P. V., K. Takegawa, M. J. Fry, J. H. Stack, M. D. Waterfield, and S. D. Emr. 1993. Science [Wash. DC]. 260:88-91). The activity of Vps34p kinase is significantly reduced by the PI 3-kinase inhibitors wortmannin, a fungal metabolite, and LY294002, a quercetin analog (Stack, J. H., and S. D. Emr. 1994. J. Biol. Chem. 269:31552-31562). We show here that at concentrations which inhibit VPS34-encoded PI 3-kinase activity, wortmannin also inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in mammalian cells with half-maximal inhibition of delivery occurring at 100 nM wortmannin. As a result of wortmannin action, newly synthesized, unprocessed cathepsin D is secreted into the media. Moreover, after accumulation in the trans-Golgi network (TGN) at 20 degrees C, cathepsin D was rapidly missorted to the secretory pathway after addition of wortmannin and shifting to 37 degrees C. At concentrations that inhibited lysosomal enzyme delivery, both wortmannin and LY294002 caused a highly specific dilation of mannose 6-phosphate receptor (M6PR)-enriched vesicles of the prelysosome compartment (PLC), which swelled to approximately 1 micron within 15 min after treatment. With increasing time, the inhibitors caused a significant yet reversible change in M6PR distribution. By 3 h of treatment, the swollen PLC vacuoles were essentially depleted of receptors and, in addition, there was a fourfold loss of receptors from the cell surface. However, M6PRs were still abundant in the TGN. These results are most consistent with the interpretation that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering with a M6PR-dependent sorting event in the TGN. Moreover, they provide evidence that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles rely on similar regulatory mechanisms.

Acridine Orange↗