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. ***
