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

高尔基体由多个隔间组成，蛋白质在这些隔间接受特定的修饰或处理。因此，它们的组成和组成对于我们理解高尔基体的功能很重要。早期隔室中的Sed5蛋白对于运输囊泡和高尔基体的融合是必不可少的。我们发现Sly1蛋白与Sed5的N-末端结合，这种结合刺激高尔基体上的Sed5与运输小泡上的Bet1形成复合体。接下来，我们发现Sly1还刺激复合体的解离，因为温度敏感的sec18和sly1突变是合成致死的，并且Sed5-Bet1复合体在sly1突变体中是稳定的。我们发现GDP-甘露糖转运体(GMT)的C-末端亲水区对于GMT在高尔基体中的定位是重要的。含有GMT C端肽的GST-融合蛋白与辅助体的β-COP亚基结合。通过检测突变多肽，赖氨酸残基的数量和位置对结合很重要，结合减少的GMT错误地定位在液泡上。利用Sed5和Tlg2的免疫吸附作用，我们分别从高尔基体早期和晚期的隔室中分离出囊泡。我们成功地在这些囊泡中鉴定了40和37种膜蛋白。甘露糖基转移酶、囊泡外壳蛋白、分类蛋白是两个小泡所特有的，而小的GTP酶则存在于两个小泡中。发现了新的膜蛋白Svp26、Tvp38、Tvp23、Tvp18、Tvp15和Gvp36。在真核生物中进化上保守的TVP蛋白之间存在蛋白质-蛋白质相互作用。

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.