Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature.
Bottom Line: Phosphatidylinositol-4-phosphate (PI4P) is produced on both the Golgi and the plasma membrane.We show here that in yeast the oxysterol-binding proteins Osh1-Osh7 are collectively needed to maintain the normal distribution of PI4P and that Osh4p is critical in this function.This reduction in PI4P is necessary for a switch in the regulation of the Sec4p exchange protein, Sec2p, from an interaction with the upstream Rab, Ypt31/32, to an interaction with a downstream Sec4p effector, Sec15p.
Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093.Show MeSH
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Mentions: Osh4p binds to both ergosterol and PI4P (Im et al., 2005; de Saint-Jean et al., 2011). A prior study demonstrated that ergosterol is important for Osh4p localization (Alfaro et al., 2011). We speculated that the polarized localization of Osh4p might also be dependent on binding to PI4P, based on PI4P's cellular distribution. To test this hypothesis, we first determined whether the localization of Osh4 is affected by the loss of enzymatic activities that synthesize PI4P. In budding yeast, there are two major PI 4-kinases responsible for PI4P synthesis. Pik1 is a Golgi-localized lipid kinase, whereas Stt4 functions at the plasma membrane (Audhya et al., 2000). Each of these two PI 4-kinases synthesizes ∼50% of the total PI4P in cells. We examined the localization of Osh4-3xGFP in pik1ts and stt4ts cells, respectively. At the permissive temperature, Osh4-3xGFP localized normally in both pik1ts cells and stt4ts cells. Of interest, upon shifting to the nonpermissive temperature for 1 h, Osh4-3xGFP appeared predominantly cytoplasmic in pik1ts cells, whereas its localization was normal in stt4ts cells (Figure 3, A and B). Subcellular fractionation results further support the proposal that Pik1p function is important for Osh4p membrane association (Supplemental Figure 2, A and B). Together these results suggest that the localization of Osh4p is controlled by the Pik1-generated pool of PI4P.
Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093.