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Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature.

Ling Y, Hayano S, Novick P - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093.

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Osh4 is present on Sec4 positive structures in cells. (A) Localization of mCherry-Sec4 and Osh4-3xGFP in cells. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose. The boxed area shows a region magnified fourfold. Arrows indicate that Osh4-3xGFP is present on cytoplasmic punctate structures. Arrowheads indicate that Osh4-3xGFP colocalizes with mCherry-Sec4 in small buds. Scale bar, 2 μm. (B) The polarized localization of Osh4-3xGFP is dependent on Sec4. Osh4-3xGFP localization in wild-type and sec4ts cells at indicated temperatures. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. Scale bar, 5 μm. (C) Quantification of wild-type cells and sec4ts cells with polarized localized Osh4-3xGFP at 25 or 37°C for 1 h. A total of 200 cells was counted for each experiment. Values indicate the percentage of cells showing bud or mother–daughter neck localization of Osh4-3xGFP. Mean and SD of three experiments.
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Figure 2: Osh4 is present on Sec4 positive structures in cells. (A) Localization of mCherry-Sec4 and Osh4-3xGFP in cells. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose. The boxed area shows a region magnified fourfold. Arrows indicate that Osh4-3xGFP is present on cytoplasmic punctate structures. Arrowheads indicate that Osh4-3xGFP colocalizes with mCherry-Sec4 in small buds. Scale bar, 2 μm. (B) The polarized localization of Osh4-3xGFP is dependent on Sec4. Osh4-3xGFP localization in wild-type and sec4ts cells at indicated temperatures. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. Scale bar, 5 μm. (C) Quantification of wild-type cells and sec4ts cells with polarized localized Osh4-3xGFP at 25 or 37°C for 1 h. A total of 200 cells was counted for each experiment. Values indicate the percentage of cells showing bud or mother–daughter neck localization of Osh4-3xGFP. Mean and SD of three experiments.

Mentions: There are seven different Osh proteins in the budding yeast S. cerevisiae. They share redundant yet essential roles in vivo, as any one is sufficient for cell growth, but collectively they are required for cell viability (Beh et al., 2001). Osh1p localizes to Golgi and nuclear–vacuolar junction sites (Levine and Munro, 2001), whereas Osh2p, Osh3p, Osh6p, and Osh7p were previously localized to ER-PM contact sites (Levine and Munro, 2001; Schulz et al., 2009; Stefan et al., 2011). To investigate which Osh protein is the master regulator of PI4P on secretory vesicles, we tagged Osh4p and Osh5p with GFP and examined their in vivo localization using fluorescence microscopy. Only Osh4-GFP displayed a good signal by fluorescence microscopy (unpublished data). To enhance the signal, we constructed a 3xGFP fusion at the C-terminus of Osh4p. We observed three major pools of Osh4-3xGFP present in wild-type cells. One pool was present on punctate structures in cytoplasm, whereas another pool was concentrated at sites of polarized secretion, including the tips of small buds and mother–daughter necks. However, the majority of Osh4-3xGFP was cytoplasmic (Figure 2A). The pool of Osh4-3xGFP found in small bud tips colocalized well with mCherry-tagged Sec4 (Figure 2A), suggesting that it is present on secretory vesicles and therefore a good candidate for the major Osh protein regulating the final stage of the secretory pathway.


Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature.

Ling Y, Hayano S, Novick P - Mol. Biol. Cell (2014)

Osh4 is present on Sec4 positive structures in cells. (A) Localization of mCherry-Sec4 and Osh4-3xGFP in cells. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose. The boxed area shows a region magnified fourfold. Arrows indicate that Osh4-3xGFP is present on cytoplasmic punctate structures. Arrowheads indicate that Osh4-3xGFP colocalizes with mCherry-Sec4 in small buds. Scale bar, 2 μm. (B) The polarized localization of Osh4-3xGFP is dependent on Sec4. Osh4-3xGFP localization in wild-type and sec4ts cells at indicated temperatures. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. Scale bar, 5 μm. (C) Quantification of wild-type cells and sec4ts cells with polarized localized Osh4-3xGFP at 25 or 37°C for 1 h. A total of 200 cells was counted for each experiment. Values indicate the percentage of cells showing bud or mother–daughter neck localization of Osh4-3xGFP. Mean and SD of three experiments.
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Figure 2: Osh4 is present on Sec4 positive structures in cells. (A) Localization of mCherry-Sec4 and Osh4-3xGFP in cells. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose. The boxed area shows a region magnified fourfold. Arrows indicate that Osh4-3xGFP is present on cytoplasmic punctate structures. Arrowheads indicate that Osh4-3xGFP colocalizes with mCherry-Sec4 in small buds. Scale bar, 2 μm. (B) The polarized localization of Osh4-3xGFP is dependent on Sec4. Osh4-3xGFP localization in wild-type and sec4ts cells at indicated temperatures. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. Scale bar, 5 μm. (C) Quantification of wild-type cells and sec4ts cells with polarized localized Osh4-3xGFP at 25 or 37°C for 1 h. A total of 200 cells was counted for each experiment. Values indicate the percentage of cells showing bud or mother–daughter neck localization of Osh4-3xGFP. Mean and SD of three experiments.
Mentions: There are seven different Osh proteins in the budding yeast S. cerevisiae. They share redundant yet essential roles in vivo, as any one is sufficient for cell growth, but collectively they are required for cell viability (Beh et al., 2001). Osh1p localizes to Golgi and nuclear–vacuolar junction sites (Levine and Munro, 2001), whereas Osh2p, Osh3p, Osh6p, and Osh7p were previously localized to ER-PM contact sites (Levine and Munro, 2001; Schulz et al., 2009; Stefan et al., 2011). To investigate which Osh protein is the master regulator of PI4P on secretory vesicles, we tagged Osh4p and Osh5p with GFP and examined their in vivo localization using fluorescence microscopy. Only Osh4-GFP displayed a good signal by fluorescence microscopy (unpublished data). To enhance the signal, we constructed a 3xGFP fusion at the C-terminus of Osh4p. We observed three major pools of Osh4-3xGFP present in wild-type cells. One pool was present on punctate structures in cytoplasm, whereas another pool was concentrated at sites of polarized secretion, including the tips of small buds and mother–daughter necks. However, the majority of Osh4-3xGFP was cytoplasmic (Figure 2A). The pool of Osh4-3xGFP found in small bud tips colocalized well with mCherry-tagged Sec4 (Figure 2A), suggesting that it is present on secretory vesicles and therefore a good candidate for the major Osh protein regulating the final stage of the secretory pathway.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093.

Show MeSH
Related in: MedlinePlus