Role of phosphatidylserine in the localization of cell surface membrane proteins in yeast.

Phosphatidylserine (PS) is a constituent of the cell membrane, being especially abundant in the cytoplasmic leaflet, and plays important roles in a number of cellular functions, including the formation of cell polarity and intracellular vesicle transport. Several studies in mammalian cells have suggested the role of PS in retrograde membrane traffic through endosomes, but in yeast, where PS is localized primarily at the plasma membrane (PM), the role in intracellular organelles remains unclear. Additionally, it is reported that polarized endocytic site formation is defective in PS-depleted yeast cells, but the role in the endocytic machinery has not been well understood. In this study, to clarify the role of PS in the endocytic pathway, we analyzed the effect of PS depletion on endocytic internalization and post-endocytic transport. We demonstrated that in cell lacking the PS synthase Cho1p (cho1Δ cell), binding and internalization of mating pheromone α-factor into the cell was severely impaired. Interestingly, the processes of endocytosis were mostly unaffected, but protein transport from the trans-Golgi network (TGN) to the PM was defective and localization of cell surface proteins was severely impaired in cho1Δ cells. We also showed that PS accumulated in intracellular compartments in cells lacking Rcy1p and Vps52p, both of which are implicated in endosome-to-PM transport via the TGN, and that the number of Snx4p-residing endosomes was increased in cho1Δ cells. These results suggest that PS plays a crucial role in the transport and localization of cell surface membrane proteins.Key words: phosphatidylserine, endocytosis, recycling, vesicle transport.

Live-cell imaging of early coat protein dynamics during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is an essential process that is mediated by the stepwise appearance or disappearance of many different proteins at the plasma membrane. In the budding yeast, these proteins are categorized into at least five modules, according to their spatiotemporal dynamics. Among them, the dynamics of proteins in the late coat module are well characterized, but those in the early coat module still remain unclear because of the lack of a suitable fluorescent marker with sufficient brightness to allow analysis. To examine the dynamics of early coat proteins, in this study we tagged four representative early coat proteins with 3GFP, and expressed them in a single cell. This cell exhibited a significant increase in the fluorescence intensity of early coat proteins relative to that of each 3GFP-tagged protein. Using this strain, we performed a detailed analysis of early coat proteins, including their precise lifetime, changes in fluorescence intensity, and motility on the plasma membrane. We found that early coat proteins move on the plasma membrane before internalization. Additionally, we expressed these 3GFP-tagged proteins in mutants with deletion of genes related to endocytosis, and found four mutants - end3Δ, las17Δ, sla2Δ, and clc1Δ- in which the lifetime of early coat proteins was markedly increased. Interestingly, deletion of the CLC1 gene dramatically reduced the internalization of early coat proteins whereas internalization of actin patches was largely unchanged, suggesting that the clc1Δ mutant might have a defect in the link between the early coat and actin modules.