| Research Abstract |
The Golgi apparatus is composed of multiple compartments, where proteins receive specific modification or processing. Therefore, their composition and constitution are important for our understanding of the function of the Golgi.
Sed5 protein in the early compartment is essential for fusion of the transport vesicles and the Golgi. We discovered that Sly1 protein binds to the N-terminus of Sed5 and that this binding stimulates the formation of complex between Sed5 on the Golgi and Bet1 on the transport vesicle. Next we found that Sly1 also stimulates dissociation of the complex because temperature-sensitive sec18 and sly1 mutations are synthetically lethal and the Sed5-Bet1 complex is stable in the sly1 mutant.
We found that the C-terminal hydrophilic region of GDP-mannose transporter (GMT) is important for the localization of GMT in the Golgi. GST-fusion protein containing the GMT C-terminal peptide binds to β-COP subunit of the coatomer. By examining mutant peptides, the number and position of lysine residues are important for the binding and GMT with reduced binding is mislocalized to the vacuole. A dynamic movement of GMT was found by examining temperature-sensitive secretion mutants.
By immunoadsorption using Sed5 and Tlg2, we isolated vesicles derived from the early and late compartments of the Golgi, respectively. We succeeded to identify 40 and 37 membrane proteins in these vesicles. Mannosyltransferases, coat proteins of vesicles, sorting proteins are specific for either vesicles whereas small GTPases are found in both vesicles. Novel membrane proteins, Svp26, Tvp38, Tvp23, Tvp18, Tvp15, and Gvp36 are discovered. Protein-protein interaction was found among Tvp proteins which are evolutionally conserved in the eukaryote.
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