Abstract #91A

Involvement of the transbilayer redistribution of plasma membrane phospholipids in spatial regulation of polarized membrane growth. Koji Saito¹, Konomi Fujimura-Kamada¹, Utako Kato², Masato Umeda², Keith G. Kozminski³, Kazuma Tanaka¹. 1) Division of Molecular Interaction, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 2) Division of Molecular Biology and Information, Institute for Chemical Science, Kyoto University, Kyoto, Japan; 3) Departments of Biology and Cell Biology, University of Virginia, Charlottesville, VA.
   In eukaryotic cells, phospholipid asymmetry across the plasma membrane (PM) is generated by ATP-driven lipid transporters or translocases, but its physiological significance remains largely unknown. The budding yeast Dnf1p and its homolog Dnf2p are putative phospholipid translocases that seem to be involved in inward phospholipid translocation across the PM. Dnf1p and Dnf2p form a complex with Lem3p, a conserved integral membrane protein, which is likely a non-catalytic subunit necessary for their proper localization. Both Lem3p-Dnf1p and Lem3p-Dnf2p are localized to the PM at a polarized growth site (e.g. bud tip). The lem3D mutant as well as the dnf1D dnf2D mutant exhibits hyper polarized growth at lower temperatures (18°C), resulting in an ellipsoidal cell shape. This phenotype was not due to a mitotic delay, because the actin cytoskeleton was still polarized to the bud tip even after nuclear division. Consistent with this, the small GTPase Cdc42p was localized to the bud tip throughout the budding process in the lem3D mutant. Staining of phosphatidylethanolamine (PE) in the outer leaflet of the PM using a biotinylated PE-binding peptide Ro revealed that PE was exposed on the cell surface at the bud tip where Cdc42p seemed to be localized in lem3D mutant cells. Interestingly, the mutation in the COOH-terminal polylysine region of Cdc42p, which has been proposed to modulate association with membranes, suppressed the hyper polarized growth of the lem3D mutant without affecting its essential function. These results suggest that changes of phospholipid asymmetry at a specific region of the PM regulate a switch from the apical to isotropic growth phase through Cdc42p in polarized membrane growth.

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