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

职业：与 ARFI 发生遗传相互作用的酵母基因的分离和表征

• 批准号: 9600835
• 负责人: Todd Graham
• 金额: $ 30万
• 依托单位: Vanderbilt University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9600835&HistoricalAwards=false
• 关键词: CAREER; Isolation; Characterization; Yeast; Genes

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. ***

96-00835 Graham 真核细胞由功能上不同的膜分隔区室组成，称为细胞器。其中，内质网（ER）和高尔基复合体形成了一个整合的膜系统，称为分泌途径。 蛋白质靶向ER，修饰，并包装成膜结合囊泡，其本身靶向多隔室高尔基体复合物的顺式表面。 高尔基复合体作为蛋白质的分选中心，也是分泌途径中脂质的分选中心。高尔基复合体含有将糖和硫核苷酸输入高尔基室池腔的转运蛋白。高尔基复合体内的转移酶催化这些组分组装成各种各样的糖蛋白、蛋白聚糖和糖脂。一旦组装，糖缀合物被分选并包装到囊泡载体中以输出到特定的亚细胞目的地。高尔基复合体的池被组织成堆叠，这些堆叠被分成顺式面（ER近端）、中间、反式和反式高尔基网络。据推测，囊泡之间的载体运输通过膜结合囊泡发生。 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。如何做到这一点，人们知之甚少。 从高尔基复合体出芽的囊泡需要一个小的三磷酸鸟苷（GTP）结合蛋白ADP-核糖基化因子（ARF）. ARF被认为与膜结合并有助于囊泡外壳成分的募集。两类囊泡外套，网格蛋白和COP I，显然需要ARF膜结合。与其他小GTP结合蛋白类似，一些效应物应该在体内调节ARF活性。ARF的这些效应因子尚不清楚。 PI将使用酿酒酵母中开发的遗传技术来鉴定与ARF相互作用的效应子。此外，这项技术可能允许发现其产物是高尔基复合体正常功能所需的基因。以前的基因筛选缺陷的突变体分泌（秒）发现很少的突变体影响蛋白质运输通过酵母高尔基复合体。新突变体的遗传筛选及其特征将作为遗传学本科实验室练习的一部分进行。 本课程的目的是让学生体验探索未知和发现未知基因的兴奋，同时学习筛选突变体和测试互补的技术。 %%% 真核细胞由功能上不同的膜分隔区室组成，称为细胞器。其中，内质网（ER）和高尔基复合体形成了一个整合的膜系统，称为分泌途径。 蛋白质靶向ER，修饰，并包装成膜结合囊泡，其本身靶向多隔室高尔基体复合物的顺式表面。 高尔基复合体作为蛋白质的分选中心，也是分泌途径中脂质的分选中心。高尔基复合体含有将糖和硫核苷酸输入高尔基室池腔的转运蛋白。高尔基复合体内的转移酶催化这些组分组装成各种各样的糖蛋白、蛋白聚糖和糖脂。一旦组装，糖缀合物被分选并包装到囊泡载体中以输出到特定的亚细胞目的地。高尔基复合体的池被组织成堆叠，这些堆叠被分成顺式面（ER近端）、中间、反式和反式高尔基网络。据推测，囊泡之间的载体运输通过膜结合囊泡发生。 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。这是如何实现的是不太了解。 从高尔基复合体出芽的囊泡需要一个小的三磷酸鸟苷（GTP）结合蛋白ADP-核糖基化因子（ARF）. ARF被认为与膜结合并有助于囊泡外壳成分的募集。两类囊泡外套，网格蛋白和COP I，显然需要ARF膜结合。与其他小GTP结合蛋白类似，一些效应物应该在体内调节ARF活性。ARF的这些效应因子尚不清楚。 PI将使用酿酒酵母中开发的遗传技术来鉴定与ARF相互作用的效应子。此外，这项技术可能允许发现其产物是高尔基复合体正常功能所需的基因。以前的基因筛选缺陷的突变体分泌（秒）发现很少的突变体影响蛋白质运输通过酵母高尔基复合体。新突变体的遗传筛选及其特征将作为遗传学本科实验室练习的一部分进行。 本课程的目的是让学生体验探索未知和发现未知基因的兴奋，同时学习筛选突变体和测试互补的技术。 ***