Cloning and characterization of genes encoding proteins involved in the terminal stage of vesicular traffic in the yeast Saccharomyces cerevisiae: Dissertation

In eukaryotic cells, the compartmentalization of biochemical reactions requires a system for proper intracellular sorting of proteins destined for the different organelles of the cell. The secretory pathway consists of membrane enclosed compartments through which proteins are transported either to the plasma membrane or to a final destination inside the cell. In the baker's yeast, Saccharomyces cerevisiae, the secretory pathway was first characterized through the use of temperature sensitive mutants which block secretion and cell surface growth at the restrictive temperature, thus causing an abnormally large intracellular pool of secretory enzymes inside the cell. The wild type copy of the yeast S. cerevisiae SEC1 gene was cloned by using its ability to restore the growth of a sec1 1 temperature sensitive mutant strain at the restrictive temperature. At this temperature, growth and secretion cease in sec1 1cells. Inside the cells, secretory vesicles accumulate, which contain fully matured secretory proteins destined for the plasma membrane or the outside of the cell. In this work it was found that SEC1 is a single copy essential gene which encodes a large protein of hydrophilic nature. Database searches revealed that two other SEC1 related genes exist in yeast: SLY1, which is needed in vesicular transport between endoplasmic reticulum and Golgi apparatus, and VPS33/SLP1, which functions in the Golgi to vacuole transport. That related proteins function in different parts of the secretory pathway suggests that biochemically similar mechanisms are used at different stages of vesicular transport. The yeast genes SSO1 and SSO2 were cloned by using their ability, when highly over expressed, to partially restore the growth of sec1 1 mutant. They suppress temperature sensitive SEC1 mutations, but not a disruption of SEC1. Together, SSO1 and SSO2 perform an essential function in vesicular transport. SSO1 and SSO2 were found to be related to two other genes in yeast. One is SED5, which is involved in the transport of secretory vesicles between the endoplasmic reticulum and Golgi apparatus. The other is PEP12, which functions in transport from the Golgi to the vacuole. These three proteins are related to mammalian syntaxin proteins, which functions at the terminal stage of the vesicular transport in neuronal cells. The finding of duplicated members of the syntaxin family within the secretory pathway in yeast reinforces the notion that components of the secretory machinery have been duplicated at least twice during evolution, when new organelles emerged. The MSO1 gene was cloned by using its ability, when overexpressed, to restore the growth of sec1-1. It encodes a small protein of 211 amino acids, which has a hydrophilic nature. The gene itself is not an essential one, but a disruption of MSO1 is lethal together with the sec1 1 mutation. Using a two hybrid analysis, I found that Mso1 protein binds to Sec1 protein. The interacting domains of the two proteins were mapped.