Randy W. Schekman - US grants

We have isolated a series of yeast secretory mutants (sec mutants) that are temperature-sensitive for cell surface growth, division, and secretion. Most of these mutants accumulate secretory proteins in an intracellular pool which can be released when cells are returned to a permissive temperature. At least 23 gene products have been implicated in the process of delivering membrane and secretory proteins to the cell surface. Electron microscope analysis has revealed three distinct membrane-bounded organelles that accumulate in different mutants. Ten of the mutants produce vesicles, nine of the mutants produce endoplasmic reticulum, and two of the mutants produce Golgi body-like structures which we have called Berkely bodies. We have also identified nine genes that contrrl the earliest steps in the cell surface growth pathway. Mutations in these genes result in a failure to synthesize active secretory protein, in spite of a normal rate of overall protein synthesis. Two of the mutants are also defective in the production of a membrane permease activity. The secretory mutants are now being used to identify some of the molecular events involved in cell-surface growth. In higher eukaryotic cells, glycoproteins are glycosylated in stages during their transit to the cell surface and thus if a secretory mutant blocks passage from one stage to another, incompletely glycosylated forms may accumulate. We have detected at least two stages in oligosaccharide assembly on the secretory enzyme invertase. The secretory mutants that accumulate endoplasmic reticulum at a nonpermissive temperature produce an incompletely glycosylated form of invertase.

Clathrin-coated vesicles and membranes have been implicated in a variety of intracellular transport processes in eukaryotic cells. The precise funciton of clathrin in cell growth and protein transport will be addressed by evaluation of yeast mutants defective in production of clathrin heavy and light subunits. Polyclonal antiserum was used to identify a clone of the yeast heavy chain gene (CHC1) expressed in a Lambdagtll genomic library. The identity of this insert was confirmed by hybrid-selected translation of heavy chain mRNA followed by immune precipitation. A fragment of the insert was then used to generate deletions of the chromosomal CHC1 locus. Surprisingly, viable deletion mutant strains were produced which have no detectable immunoreactive heavy chain, and which produce vesicles that also lack clathrin light chain. Although clathrin deletion mutant cells grow slowly, secretion of the glycoprotein invertase is nearly normal. Deletion mutant cells will now be examined for the rate and fidelity of transport and sorting of proteins to various destinations. Structural analogues of clathrin will be sought using sensitive nucleic acid hybridization procedures and by examining alternative associations formed with clathrin light chain in heavy chain deletion mutant cells. Cloning and deletion analysis of the light chain gene will be used to detect any independent role that light chains may play in protein transport.

Polypeptide translocation from the cytosol into the lumen of the endoplasmic reticulum represents the first step in the secretory pathway, and the only event that requires passage of a hydrophilic protein through a lipid bilayer. Recent years have seen a dramatic convergence of observations that suggest a fundamental conservation of the mechanism of protein translocation in prokaryotes and eukaryotes. Work conducted on this grant since the last renewal has led to the discovery of a set of ER membrane proteins and cytosolic hsc7Os that constitute essential elements of the translocation apparatus. One of these molecules, Sec6lp, provides a link between core elements of the E. coli and mammalian polypeptide translocase. Although many of the membrane, cytosolic, and lumenal proteins required for polypeptide import into the ER have now been defined, the exact mechanism of penetration remains obscure. In the previous grant period we developed a method to solubilize yeast membranes and reconstitute translocation in proteoliposomes. One membrane protein complex that contains Sec63p, BiP, and two new Sec proteins was isolated. Additional membrane proteins that are required for translocation are now being isolated with the goal being a fully purified system with which to investigate the nature of the putative channel. Crucial questions of subunit stoichiometry, interactions among the Sec proteins, regulation of channel assembly, and capacity to translocate small molecules will be addressed using Sec proteins isolated from wild type and mutant cells. With a purified set of translocation Sec proteins, we will examine the components that are responsible for signal peptide binding and that are involved in completion of protein import. We have proposed that members of the Sec63p complex interact with the signal peptide portion of presecretory proteins and deliver them to the Sec6lp channel. A subreaction that measures binding of isolated signal peptides to proteoliposomes or soluble membrane proteins is described. A partial reaction that measures BiP-dependent completion of polypeptide transport through the Sec6lp channel will be reproduced in proteoliposomes and in detergent solution. The selective-role of hsc7Os will be examined in the proteoliposome reaction using chimeric molecules made of cytosolic hsc70 and BiP. We hope to define domains of these molecules that confer topological specificity.