We have identified an elaborate genetic interaction among three genes (BOS1, BET1 and SEC22) required for the transit of proteins from the lumen of the ER to the Golgi complex in the yeast Saccharomyces The goal of our research is to define the nature of these interactions by performing a detailed analysis of these genes and their products. We have developed an assay that reconstitutes transport from the ER through the Golgi complex in yeast (Ruohola et al., 1988). Recently, we have used this assay to identify a functional vesicular intermediate in transit from the ER to the Golgi apparatus. The isolation of this intermediate has enabled us to order events with respect to vesicle formation. The Bos1, Bet1 and Sec22 proteins may be required for the budding of vesicles from the ER or the binding and fusion of vesicular carriers to the acceptor Golgi compartment. The experiments described in this proposal will utilize molecular genetics, cell fractionation and the in vitro assay to address this possibility and to determine the function of these gene products. 1. We will sequence the SEC22 gene and obtain antibody to the protein encoded by this gene. The anti-Sec22 antibody will be used to assess the intracellular location of the Sec22 protein. 2. DNA sequence analysis predicts that the BOS1 and BET1 genes encode putative membrane proteins. We will use antibodies prepared to these gene products to ascertain their intracellular location and topology. We will also determine if Bos1 and Bet1 are glycosylated proteins that traverse the secretory pathway or a portion of this pathway. 3. Antibodies to the Bos1, Bet1, and Sec22 proteins will be used to define the interactions observed among these genes products. These experiments will reveal whether the Bos1, Bet1 and Sec22 proteins are components of a complex. Alternatively, these proteins may functionally interact on the same pathway or parallel pathways. In addition, we will identify soluble proteins that may interact with the hydrophilic domain of the Bos1 and Bet1 proteins. 4. We will screen for homologues of the BOS1, BET1 and SEC22 genes in mammalian cells. 5. We will determine if antibodies prepared to the Bos1, Bet1 and Sec22 proteins block vesicle formation or subsequent stages of transport in vitro.
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