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We have previously identified three mammalian Sec1/ Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin-1A, -2, and -3, while Munc-18c binds only to syntaxin-2 and -4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin-1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Munc-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP-2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes.In adipose tissue and muscle, the glucose transporter isoform 4, GLUT-4, is translocated from an intracellular vesicular pool to the cell surface in response to insulin (1, 2), a process that plays a major role in whole body glucose homeostasis. To understand the molecular mechanisms governing this vesicular transport system, it will be necessary to identify and characterize the individual components of the trafficking machinery. The SNARE hypothesis (reviewed in Ref.3) provides a working model for studies of vesicle targeting and fusion in adipocytes. Vesicle-associated membrane protein VAMP or synaptobrevin (v-SNAREs) 1 present on the transport vesicle and syntaxin (t-SNAREs) on the acceptor membrane form a complex, which also includes the synaptosomal-associated protein-25 (SNAP-25), soluble N-ethylmaleimide-sensitive factor attachment protein (␣-SNAP), and N-ethylmaleimide-sensitive factor (NSF). This complex may facilitate the docking and/or fusion of distinct membrane compartments, the specificity being provided by the pairing of unique v-and t-SNAREs at different loci throughout the cell. Each of the SNAREs belong to large gene families. For example, in mammalian cells seven different syntaxins (4, 5), ...

Vpr is a virion-associated protein of human immunodeficiency type 1 (HIV-1) whose function in acquired immunodeficiency syndrome (AIDS) has been uncertain. Employing the yeast Saccharomyces cereviswae as a model to examine the effects of HIV-1 auxiliary proteins on basic cellular functions, we found that the vpr gene caused cell growth arrest and structural defects indicated by osmotic sensitivity and gross cell enlargement. Production of various domains by gene expression showed that this effect arose from within the carboxyl-terminal third of the Vpr protein and implicated the sequence HFRIGCRHSRIG, containing two

We have previously shown that expression of HIV-1 vpr in yeast results in cell growth arrest and structural defects, and identified a C-terminal domain of Vpr as being responsible for these effects in yeast. In this report we show that recombinant Vpr and C-terminal peptides of Vpr containing the conserved sequence HFRIGCRHSRIG caused permeabilization of CD4+ T lymphocytes, a dramatic reduction of mitochondrial membrane potential and finally cell death. Vpr and Vpr peptides containing the conserved sequence rapidly penetrated cells, co-localized with the DNA, and caused increased granularity and formation of dense apoptotic bodies. The above results suggest that Vpr treated cells undergo apoptosis and this was confirmed by demonstration of DNA fragmentation by the highly sensitive TUNEL assay. Our results, together with the demonstration of extracellular Vpr in HIV infected individuals, suggest the possibility that extracellular Vpr could contribute to the apoptotic death and depletion of bystander cells in lymphoid tissues during HIV infection.

Virus-specific or group-specific antibody probes to potyviruses can be produced by targeting the immune response to the virus-specific, N-terminal region of the capsid protein (29-95 amino acids depending on the virus) or to the conserved core region (216 amino acids) of the capsid protein, respectively. Immunochemical analysis of overlapping, synthetic octapeptides covering the capsid protein of the Johnsongrass strain of Johnsongrass mosaic virus (JGMV-JG) has delineated the peptide sequences recognized by five polyclonal rabbit antisera and two mouse monoclonal antibodies (mAbs). The antibodies characterized were (i) three virus-specific rabbit polyclonal antisera and one virus-specific mouse mAb (1/25) raised against native virus particles, (ii) one polyclonal antiserum raised against trypsin-derived core particles of JGMV-JG, (iii) one group-specific polyclonal antiserum raised against the denatured, truncated coat protein from trypsinderived core particles of JGMV-JG, and (iv) one group-specific mouse mAb (1/16) raised against native virus particles. The two epitopes seen by mAb 1/25 occurred at residues 18-27 and 43-52 and overlapped with the two major epitopes seen by the virus-specific polyclonal antiserum. The group-specifilc epitope seen in JGMV-JG by mAb 1/16 was also recognized strongly in potato virus Y. the type member of the potyvirus group. The multiple epitopes seen by the cross-reactive polyclonal antisera were distributed across the entire core region of the coat protein and their relative antibody binding responses varied between JGMV-JG, potato virus Y, and six other distinct potyviruses.Plant diseases are estimated to be responsible for economic losses worldwide of $60 billion per annum (1). The most important pathogens are fungi, with plant viruses the second most important group of infectious agents. Of the 28 plant virus groups or families the potyvirus group is the largest. It contains at least 175 independent members and accounts for about 30% of all viruses known to infect plant species around the world (2, 3). Potyvirus particles are flexuous rods, 729-900 nm long and -11 nm in diameter, and consist of up to 2000 subunits of a single protein species (4).Successful control and eradication of plant virus infections is dependent on the availability of simple, reliable methods for plant virus detection and identification. To date, this has been difficult to achieve for potyviruses due to the large size of the group, the vast variation between members, and the lack of satisfactory taxonomic parameters to distinguish independent viruses from related strains (2-4). Thus, there is a real need to evaluate new criteria for the identification and classification of potyviruses.During investigations on the structural characterization of the coat proteins of potyviruses we made the following observations that have implications for potyvirus detection and classification: (i) distinct potyviruses exhibit coat protein sequence homology of 38-71% with major differences in the length (29-95 re...