Functional Studies of Vesicular Transport in Yeast

Barmark, G. 2005. Functional studies of vesicular transport in yeast. Doctor’s dissertation. ISSN 1652-6880, ISBN 91-576-6909-0 A basic feature of eukaryotic cells is their compartmental organisation. To maintain this cellular order, a strictly regulated intracellular transport is essential. The compartments are defined by their lipid membranes and the transport between different compartments is therefore mediated by lipid vesicles. The yeast Saccharomyces cerevisiae is a very suitable model organism for studies of intracellular transport. In order to find new yeast genes involved in intracellular transport, a screen was performed using a knockout yeast strain collection and two drugs known to interfere with intracellular transport, monensin and brefeldin A. In an initial screen, seven new genes were found (MON1-2 and BRE1-5) that caused an increased sensitivity to either drug when knocked out. When the monensin screen was extended to the entire yeast genome, a total of 63 sensitive strains were found. Most of the encoded proteins are known to be involved in post-Golgi transport. Some of the 63 monensin-sensitive knockout mutants showed synthetic interactions with a vma1 knockout mutant, ranging from complete synthetic lethality to reduced growth. These interactions were specific for genes involved in retrograde transport. One of the new genes found in the screen, MON1, is involved in transport to the vacuole. A mon1 deletion strain is impaired in maturation of proaminopeptidase both during starvation and during nutrient-rich conditions, indicating that Mon1p is involved in both the autophagic and the cytoplasm-to-vacuole targeting pathways. A GFP-Mon1p fusion protein localizes to the cytosol, and to punctate structures in the cytosol. Furthermore, the autophagosomal marker protein GFP-Aut7p accumulates in mon1 deleted cells at punctate structures near the vacuole. The two syntaxins, Sso1p and Sso2p, are involved in the fusion of vesicles to the plasma membrane, where they can functionally replace each other. However, the SSO1 gene also has an essential function in meiosis, which SSO2 cannot provide. Gene fusions between SSO1 and SSO2 were used to map this meiosis-specific function to two short elements within the 3'-untranslated region of the SSO1 gene.