UCSF

The plasma membrane is a highly dynamic organelle controlling the traffic of molecules, nutrients, and ions into and out of the cell according to need. We discovered a novel protein complex required for proper membrane integrity and marks sites of endocytosis, termed `eisosome'. Eisosomes are static assemblies that distribute uniformly in a punctate pattern under the plasma membrane. We currently do not understand the mechanism of formation by which they are inherited to maintain stability, uniform size, distribution and organization. We explored the rules of eisosome formation and distribution focusing on the role of Pil1, one of the main components, in their formation and organization. In addition, we conducted a genomic-wide visual screen to identify genes required for proper eisosome organization. Eisosome formation happens de novo in daughter cells in a polarized fashion with an initial lag phase. Lowering Pil1 levels reduces eisosome density to maintain a minimal size while increased Pil1 levels results in larger eisosomes while maintaining the set density. This is not true when Pil1 is removed from cell cycle control. We show that the temporal expression level of Pil1 during G2/M phase of the cell cycle controls eisosome size and set density.

The genome-wide screen identified two novel eisosome components, Nce102 and Pen1. Deletion of either gene results in reduced eisosome assembly and increased Pil1 localization to the cytoplasm but their modes of action are different. Nce102 moves into and out of eisosomes in response to sphingolipid levels, and found to negatively regulate Pkh signaling and eisosome assembly. Nce102 deletion causes an increase in Pil1 phosphorylation mediated by the Pkh kinases and a nonphosphorylatable mutant of Pil1 is resistant to it. Pen1 was found to be an eisosome nucleator, its deletion results in a delayed aberrant eisosome formation and plasma membrane deformities. Its deposition in small buds lacks a lag phase and it precedes Pil1. Its association to the membrane is independent of Pil1, but Pil1 is needed to stabilize its localization to eisosomes. In addition, Pen 1 overexpression results in overnucleated long shaped eisosomes and a mutation on the second phenylalanine rescues this phenotype.