The role of endocytosis during sporulation in Saccharomyces cerevisiae.
Masayo Morishita (1), JoAnne Engebrecht (2)
(1) Molecular & Cellular Biology, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA; (2) Molecular & Cellular Biology, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA
During sporulation in Saccharomyces cerevisiae, bilayered prospore membranes (PSMs) are formed by the fusion of vesicles at spindle pole bodies. Vesicles are continuously transported to PSMs for the deposition of proteins responsible for the construction of spore walls, which are composed of four layers. Membrane trafficking plays an important role in supplying vesicles for these processes. The endocytosis-deficient mutant, end3, produced immature spores, suggesting END3 -mediated endocytosis is important for spore wall formation. End3p-GFP co-localized with actin structures at cell and spore peripheries in vegetative and sporulating cells. Correspondingly, the actin cytoskeleton appeared aberrant during sporulation in end3. The early stages of spore formation (PSM formation, enzymatic activity of the major yeast phospholipase D required for PSM formation, and the inner layers of spore walls) are not impaired by END3 ablation. However, formation of the outer layers, chitosan and dityrosine, was severely affected in end3. As endocytosis was only partially blocked during sporulation in the end3 mutant, we are using chemical genetics to control the kinase activity of an analogue-sensitive mutant Prk1p, which is required for actin organization and endocytosis, by adding the inhibitor, 1NA-PP1, and monitoring sporulation. Analysis of this mutant in the presence of inhibitor should uncover the role of endocytosis throughout the sporulation program.