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: PI4P inhibits the interaction between Sec2p and Sec15p both in vivo and in vitro (Mizuno-Yamasaki et al., 2010); therefore we investigated whether this interaction is also controlled by Osh4p in vivo. We tagged Sec2p with 3xGFP and Sec15p with 13myc in wild-type, osh4Δ, and sac1Δ cells. We performed coimmunoprecipitation experiments, pulling down Sec2-3xGFP with the anti-GFP antibody and then probing for Sec15p with anti-myc antibody. Consistent with the microscopy data, we observed that in both osh4Δ and sac1Δ cells, less Sec15-13myc was coimmunoprecipitated than with wild-type cells (Figure 6A). Quantification of the coprecipitation experiment revealed that the interaction between Sec2p and Sec15p was roughly threefold lower on average in osh4Δ and sac1Δ cells than with wild-type cells (Figure 6B). These data further support the model that Osh4p negatively regulates PI4P levels of secretory vesicles and high levels of PI4P in osh4 Δ cells inhibits the interaction between Sec2p and Sec15p.
Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093.