Probing the universal role of Sec1/Munc18 proteins by mutagenesis of yeast Sec1

The Sec1/Munc18 (SM) family of proteins is essential for intracellular vesicle trafficking in eukaryotic cells. Sec1, the SM protein at the yeast plasma membrane, is required for membrane fusion leading to secretion and cell growth. Fusion of exocytic vesicles targeted to the plasma membrane occurs in two stages. The vesicle is first tethered to the membrane by a protein complex called the exocyst. Essential membrane-associated proteins known as the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) located on both the plasma membrane and the vesicle assemble together to form a stable four-helix bundle called the SNARE complex. In the second step, Sec1 binds to the assembled SNARE complex and there is fusion of the two membranes. Sec1 has been shown to bind the SNARE complex, but the exact role and mechanism of Sec1 function remains unknown. In this study, site-directed mutagenesis was used to investigate the significance of highly conserved salt bridges in SM proteins and define the SNARE complex binding surface on Sec1 with data from random mutants studied by Yi-Shan Cheng and Jenna Hutton. In addition, Sec1 mutants with a novel phenotype were generated that may support a role for Sec1 in vesicle tethering.M.S.Includes bibliographical references (p. 64-68)