Screening for Exo84 Gain-of-Function Mutants that Mimic Activation of the Exocyst Complex by Sro7/77

Polarized cells, such as the yeast strain Saccharomyces cerevisiae, manifest their cell polarity through the transport, tethering, and fusion of post-Golgi vesicles to specific regions of the cell membrane. Tethering is achieved in part by activation of an evolutionarily conserved complex of eight proteins called the exocyst. Recent evidence suggests that the S. cerevisiae exocyst is partially activated through the interaction of lethal giant larvae protein homolog, Sro7/77, and a component of the exocyst, Exo84. However, the function and mechanism of this activation is unknown. Our hypothesis was that gain-of-function Exo84 mutants can mimic constitutive activation by Sro7/77, and thereby suppress deleterious phenotypes caused by disruption of Sro7/77 and Exo84 interactions. To test this hypothesis, a library of randomly mutagenized EXO84 alleles was generated and screened at cold temperatures in yeast that were cold-sensitive due to sro7/77 deletions. Although 15 suppressors were identified, none of the extracted EXO84 alleles were shown to be the cause of suppression, suggesting that the sro7/77 deletion causes too severe a phenotype for a mutated EXO84 allele alone to rescue. Consequently, the screen was performed again at restrictive temperatures in a strain of yeast where exocyst activation by Sro7/77 is indirectly disrupted, through the mutation of an upstream regulator, Sec2. The mutated strain, sec2-41, has been shown to exhibit exocytotic defects and causes temperature sensitivity as a result. From this screen, 17 suppressors were identified and one EXO84 allele was shown to suppress temperature sensitivity through the replacement of leucine with tryptophan at amino acid 365. This mutation may give rise to suppression by relaxing the normally tight structure of Exo84 and the proteins surrounding it in the exocyst complex, thereby opening up the Sec15 binding site to Sec4 and constitutively activating the exocyst. Future directions include determining other exocytotic defects suppressed by the allele, as well as biochemical examinations to understand how the mutation gives rise to suppression of sec2-41. Because these exocytotic proteins are conserved evolutionarily between yeast and humans, understanding these mechanisms is informative for cell polarity establishment in human cells.Bachelor of Scienc