MOLECULAR GENETICS OF INTRACELLULAR PROTEIN TRANSPORT

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DESCRIPTION: The membrane-bounded organelles along the secretory pathway are responsible for delivering newly made protein and membrane to the cell surface. At each of the steps on the pathway, protein (cargo) is packaged into vesicles that bud from the membrane of the donor organelle. Fusion of these vesicles with the appropriate target membrane delivers this cargo to the next organelle in the pathway. The applicant proposes experiments in the yeast, Saccharomyces cerevisiae, to provide molecular explanations for vesicle construction at the ER membrane, cargo selection and packaging into these vesicles. Previous work from the applicant has demonstrated specific binding interactions between the proteins that are part of the coat that encapsulates the vesicles, known as COPII, that carry protein from the ER to the Golgi apparatus, and they have mapped the regions in each partner protein that are essential for the interactions. Those findings suggest that the large membrane-bound protein Sec16 is the scaffold onto which the coat is constructed from soluble protein complexes. To elucidate the mechanism of coat assembly, individual protein-protein associations will be assayed and used to test how each association depends on the others. The role of the small GTPase Sar1p in assembly of the COPII complex will also be evaluated. Assemblies of purified proteins will be examined by electron microscopy to see how the biochemical interactions generate coat structures. The applicant has recently discovered several new genes that determine which cargo molecules are packaged into vesicles and the overall selectivity of that packaging. Experiments are proposed to probe the mechanism(s) of selection and their relation to vesicle coat formation. The molecular mechanisms underlying vesicular transport appear to be the same in yeast and mammals. By studying this process in yeast the full power of molecular genetics and biochemistry can be used to identify the fundamental mechanisms and key gene products that control secretion. Potential applications to disease include Cystic Fibrosis, pulmonary emphysema, and ways to challenge the growth of membrane enveloped viruses and possibly the uncontrolled growth of tumor cells.