CAREER: Isolation and Characterization of Yeast Genes That Genetically Interact with ARFI

96-00835格雷厄姆真核细胞由功能不同的膜分隔的隔间组成，称为细胞器。其中，内质网(ER)和高尔基复合体形成一个完整的膜系统，称为分泌途径。蛋白质被靶向内质网，被修饰，并被包装成膜结合的囊泡，这些囊泡本身靶向多隔室高尔基复合体的顺式表面。高尔基复合体在分泌途径中既是蛋白质的分选中心，也是脂质的分选中心。高尔基复合体含有转运蛋白，可将糖和硫核苷酸输入高尔基体隔间池的管腔。高尔基复合体中的转移酶催化这些成分组装成一系列不同的糖蛋白、蛋白多糖和糖脂。一旦组装好，糖偶联物就会被分拣并包装成泡囊载体，然后出口到特定的亚细胞目的地。高尔基复合体的小池被组织成堆叠，分为顺面(内质网近端)、内侧、反式和反式高尔基体网。据推测，脑池之间的媒介运输是通过膜结合的小泡进行的。当脂质和蛋白质流经高尔基复合体时，高尔基复合体本身必须保持其完整性。对于这一点是如何实现的，人们知之甚少。来自高尔基复合体的囊泡的萌发需要一个小的三磷酸鸟苷(GTP)结合蛋白ADP-核糖化因子(ARF)。ARF被认为与膜结合，并有助于囊泡被膜成分的募集。有两类囊泡被膜，即网状蛋白和CoP I，显然需要ARF才能与膜结合。通过类似于其他小的GTP结合蛋白，几个效应器应该可以在体内调节ARF的活性。目前尚不清楚ARF的这些效应器。PI将使用在酿酒酵母中开发的基因技术来识别与ARF相互作用的效应器。此外，这项技术可能会发现高尔基复合体正常运作所需的基因。以前对分泌物缺陷突变体(SEC)的遗传筛选发现，很少有突变体影响蛋白质通过酵母高尔基复合体的运输。新突变体的遗传筛选及其特征将作为本科生遗传学实验室练习的一部分进行。本课程旨在让学生体验探索未知和发现未知基因的兴奋，同时学习筛选突变和测试互补的技术。真核细胞由功能上不同的膜分隔的隔间组成，称为细胞器。其中，内质网(ER)和高尔基复合体形成一个完整的膜系统，称为分泌途径。蛋白质被靶向内质网，被修饰，并被包装成膜结合的囊泡，这些囊泡本身靶向多隔室高尔基复合体的顺式表面。高尔基复合体在分泌途径中既是蛋白质的分选中心，也是脂质的分选中心。高尔基复合体含有转运蛋白，可将糖和硫核苷酸输入高尔基体隔间池的管腔。高尔基复合体中的转移酶催化这些成分组装成一系列不同的糖蛋白、蛋白多糖和糖脂。一旦组装好，糖偶联物就会被分拣并包装成泡囊载体，然后出口到特定的亚细胞目的地。高尔基复合体的小池被组织成堆叠，分为顺面(内质网近端)、内侧、反式和反式高尔基体网。据推测，脑池之间的媒介运输是通过膜结合的小泡进行的。当脂质和蛋白质流经高尔基复合体时，高尔基复合体本身必须保持其完整性。如何实现这一点还没有得到很好的理解。来自高尔基复合体的囊泡的萌发需要一个小的三磷酸鸟苷(GTP)结合蛋白ADP-核糖化因子(ARF)。ARF被认为与膜结合，并有助于囊泡被膜成分的募集。有两类囊泡被膜，即网状蛋白和CoP I，显然需要ARF才能与膜结合。通过类似于其他小的GTP结合蛋白，几个效应器应该可以在体内调节ARF的活性。目前尚不清楚ARF的这些效应器。PI将使用在酿酒酵母中开发的基因技术来识别与ARF相互作用的效应器。此外，这项技术可能会发现高尔基复合体正常运作所需的基因。以前对分泌物缺陷突变体(SEC)的遗传筛选发现，很少有突变体影响蛋白质通过酵母高尔基复合体的运输。新突变体的遗传筛选及其特征将作为本科生遗传学实验室练习的一部分进行。本课程旨在让学生体验探索未知和发现未知基因的兴奋，同时学习筛选突变和测试互补的技术。***

96-00835 Graham Eukaryotic cells consist of functionally distinct membrane-delimited compartments called organelles. Of these, the endoplasmic reticulum (ER) and the Golgi complex form an integrated system of membranes called the secretory pathway. Proteins are targeted to the ER, modified, and are packaged into membrane-bound vesicles that are themselves targeted to the cis face of the multi-compartment Golgi complex. The Golgi complex acts as a sorting center for proteins as well as for lipids in the secretory pathway. The Golgi complex contains porters that import sugar and sulfur nucleotides into the lumen of the cisternae of the Golgi compartments. Transferases within the Golgi complex catalyze the assembly of these components into a diverse menagerie of glycoproteins, proteoglycans, and glycolipids. Once assembled, the glycoconjugates are sorted and packaged into vesicular carriers for export to specific subcellular destinations. The cisternae of the Golgi complex are organized into stacks that are divided into a cis face (ER proximal), medial, trans, and trans Golgi network. It is presumed that vectorial transport between the cisternae occurs through membrane bound vesicles. While lipids and proteins flow through the Golgi complex, the Golgi complex itself must maintain its integrity. How this is achieved is poorly understood. Budding of vesicles from the Golgi complex requires a small guanosine triphosphate (GTP) binding protein ADP- ribosylation factor (ARF). ARF is posited to associate with membranes and aid in the recruitment of vesicle coat components. Two classes of vesicles coat, clathrin and COP I, apparently require ARF for membrane binding. By analogy to other small GTP-binding proteins, several effectors should modulate ARF activity in vivo. These effectors for ARF are as yet unknown. The PI will use genetic techniques developed in Saccharomyces cerevisiae to identify effectors that interact with ARF. In addition, this technique may allow for the discovery of genes whose products are required for the proper functioning of the Golgi complex. Previous genetic screens for mutants defective in secretion (sec) have uncovered very few mutants effecting protein transport through the yeast Golgi complex. The genetic screen for new mutants and their characterization will be carried out as part of an undergraduate laboratory exercise in genetics. This course will be designed to allow the students to experience the excitement of probing the unknown and of discovering unknown genes, while learning techniques in screening mutants and in testing for complementation. %%% Eukaryotic cells consist of functionally distinct membrane-delimited compartments called organelles. Of these, the endoplasmic reticulum (ER) and the Golgi complex form an integrated system of membranes called the secretory pathway. Proteins are targeted to the ER, modified, and are packaged into membrane-bound vesicles that are themselves targeted to the cis face of the multi-compartment Golgi complex. The Golgi complex acts as a sorting center for proteins as well as for lipids in the secretory pathway. The Golgi complex contains porters that import sugar and sulfur nucleotides into the lumen of the cisternae of the Golgi compartments. Transferases within the Golgi complex catalyze the assembly of these components into a diverse menagerie of glycoproteins, proteoglycans, and glycolipids. Once assembled, the glycoconjugates are sorted and packaged into vesicular carriers for export to specific subcellular destinations. The cisternae of the Golgi complex are organized into stacks that are divided into a cis face (ER proximal), medial, trans, and trans Golgi network. It is presumed that vectorial transport between the cisternae occurs through membrane bound vesicles. While lipids and proteins flow through the Golgi complex, the Golgi complex itself must maintain its integrity. How this is achieved is poorly u nderstood. Budding of vesicles from the Golgi complex requires a small guanosine triphosphate (GTP) binding protein ADP- ribosylation factor (ARF). ARF is posited to associate with membranes and aid in the recruitment of vesicle coat components. Two classes of vesicles coat, clathrin and COP I, apparently require ARF for membrane binding. By analogy to other small GTP-binding proteins, several effectors should modulate ARF activity in vivo. These effectors for ARF are as yet unknown. The PI will use genetic techniques developed in Saccharomyces cerevisiae to identify effectors that interact with ARF. In addition, this technique may allow for the discovery of genes whose products are required for the proper functioning of the Golgi complex. Previous genetic screens for mutants defective in secretion (sec) have uncovered very few mutants effecting protein transport through the yeast Golgi complex. The genetic screen for new mutants and their characterization will be carried out as part of an undergraduate laboratory exercise in genetics. This course will be designed to allow the students to experience the excitement of probing the unknown and of discovering unknown genes, while learning techniques in screening mutants and in testing for complementation. ***