Study on the Molecular Mechanism to Control the Function of the Yeast Golgi Apparatus

高尔基体由多个隔室组成，蛋白质在其中接受特定的修饰或加工。因此，它们的组成和构成对于我们理解高尔基体的功能是重要的。Sed 5蛋白在早期隔室中对于运输囊泡和高尔基体的融合是必不可少的。我们发现Sly 1蛋白结合到Sed 5的N-末端，并且这种结合刺激高尔基体上的Sed 5和运输囊泡上的Bet 1之间形成复合物。我们还发现Sly 1也刺激复合物的解离，因为温度敏感的sec 18和sly 1突变是合成致死的，并且Sed 5-Bet 1复合物在sly 1突变体中是稳定的。我们发现GDP-甘露糖转运蛋白（GMT）的C-末端亲水区域对于GMT在高尔基体中的定位是重要的。含有GMT C末端肽的GST融合蛋白与外壳体的β-COP亚基结合。通过检查突变肽，赖氨酸残基的数量和位置对于结合是重要的，并且结合减少的GMT错误定位于液泡。通过检测温度敏感性分泌突变体发现GMT的动态运动。通过使用Sed 5和Tlg 2的免疫吸附，我们分离了分别来自高尔基体的早期和晚期隔室的囊泡。我们成功地确定了40和37膜蛋白在这些囊泡。甘露糖基转移酶、囊泡的外壳蛋白、分选蛋白对任一囊泡都是特异性的，而小GTP酶在两种囊泡中均被发现。发现了新的膜蛋白Svp 26、Tvp 38、Tvp 23、Tvp 18、Tvp 15和Gvp 36。Tvp蛋白在真核生物中是进化保守的，它们之间存在蛋白质间的相互作用。

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.