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我们了解高尔基体的功能是很重要的。早期腔室中的Sed5蛋白对于运输囊泡和高尔基体的融合至关重要。我们发现，Sly1蛋白与Sed5的n端结合，这种结合刺激了高尔基体上Sed5和运输囊泡上Bet1之间复合物的形成。接下来我们发现，由于温度敏感的sec18和Sly1突变是合成致死的，并且Sed5-Bet1复合物在Sly1突变体中是稳定的，因此，Sly1也刺激了复合物的解离。我们发现gdp -甘露糖转运体（GMT）的c端亲水区对GMT在高尔基体中的定位很重要。含有GMT c端肽的gst融合蛋白与涂层的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结合很重要，并且结合减少的GMT错误定位于液泡。通过检查对温度敏感的分泌物突变体，发现GMT的动态运动。通过免疫吸附Sed5和Tlg2，我们分别从高尔基体的早期和晚期区室分离出囊泡。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、分选蛋白对两个囊泡都有特异性，而在两个囊泡中都发现了小的gtp酶。发现了新的膜蛋白Svp26、Tvp38、Tvp23、Tvp18、Tvp15和Gvp36。在真核生物进化保守的Tvp蛋白之间发现了蛋白间的相互作用。

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.