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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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Osh proteins regulate PI4P distribution in cells. (A) Localization of GFP-PHFAPP1 in osh1-7Δ /CEN OSH4 cells or osh1-7Δ /CEN osh4ts 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. Cells shown are representative of >100 cells observed. Note that 6 and 55% of osh1-7Δ/CEN osh4ts cells showed polarized localization of GFP-PHFAPP1 at 25 and 37°C, respectively, and 6 and 0% of osh1-7Δ/CEN OSH4 cells showed polarized localization of GFP-PHFAPP1 at 25 and 37°C, respectively. Scale bar, 5 μm. (B) Localization of GFP-PHFAPP1 and FM4-64 in osh1-7Δ/CEN osh4ts cells at 37°C. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. FM4-64 was added to cells and kept on ice to label the plasma membrane. Scale bar, 5 μm. (C) The PI4P probe GFP-2xPHOsh2 accumulates at polarized growth sites in sec6ts cells at the nonpermissive temperature. Wild-type and sec6ts cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 15 min. Cells shown are representative of >100 cells observed. Scale bar, 5 μm.
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Figure 1: Osh proteins regulate PI4P distribution in cells. (A) Localization of GFP-PHFAPP1 in osh1-7Δ /CEN OSH4 cells or osh1-7Δ /CEN osh4ts 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. Cells shown are representative of >100 cells observed. Note that 6 and 55% of osh1-7Δ/CEN osh4ts cells showed polarized localization of GFP-PHFAPP1 at 25 and 37°C, respectively, and 6 and 0% of osh1-7Δ/CEN OSH4 cells showed polarized localization of GFP-PHFAPP1 at 25 and 37°C, respectively. Scale bar, 5 μm. (B) Localization of GFP-PHFAPP1 and FM4-64 in osh1-7Δ/CEN osh4ts cells at 37°C. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. FM4-64 was added to cells and kept on ice to label the plasma membrane. Scale bar, 5 μm. (C) The PI4P probe GFP-2xPHOsh2 accumulates at polarized growth sites in sec6ts cells at the nonpermissive temperature. Wild-type and sec6ts cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 15 min. Cells shown are representative of >100 cells observed. Scale bar, 5 μm.

Mentions: The phosphoinositide PI4P is critical for the localization of a number of proteins involved in polarized secretion, including Sec2p (Mizuno-Yamasaki et al., 2010). Here we investigate how the level of PI4P is regulated on secretory vesicles. Studies from several groups have demonstrated that the oxysterol-binding proteins (Osh proteins) control the level and intracellular distribution of PI4P in the budding yeast Saccharomyces cerevisiae (LeBlanc and McMaster, 2010; Stefan et al., 2011). To begin our analysis of the role of Osh proteins in PI4P distribution, we used cells lacking all endogenous Osh proteins and solely expressing a temperature-sensitive osh4ts allele. Consistent with a prior report (Stefan et al., 2011), we observed that the PI4P probe GFP-PHFAPP1, consisting of the PH domain from the FAPP1 protein fused to green fluorescent protein (GFP), shifted from the Golgi apparatus to the plasma membrane in the osh1-7Δ osh4ts cells after a temperature shift to 37°C (Figure 1A). In addition, there was significantly more GFP-PHFAPP1 at polarized secretory sites, such as the tips of small buds and the necks of large-budded cells, in osh1-7Δ osh4ts cells at the nonpermissive temperature (Figure 1A). These GFP-PHFAPP1–positive structures could represent either secretory vesicles containing abnormally high levels of PI4P or an accumulation of PI4P produced at the plasma membrane. To differentiate these two possibilities, we labeled the plasma membrane with the lipophilic dye FM4-64. We noticed that the GFP-PHFAPP1 positive structures accumulated in the small buds of osh1-7Δ osh4ts cells were clearly internal to the FM4-64–labeled plasma membrane (Figure 1B). These results suggest that one or more Osh proteins are needed to reduce the level of PI4P on secretory vesicles.


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)

Osh proteins regulate PI4P distribution in cells. (A) Localization of GFP-PHFAPP1 in osh1-7Δ /CEN OSH4 cells or osh1-7Δ /CEN osh4ts 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. Cells shown are representative of >100 cells observed. Note that 6 and 55% of osh1-7Δ/CEN osh4ts cells showed polarized localization of GFP-PHFAPP1 at 25 and 37°C, respectively, and 6 and 0% of osh1-7Δ/CEN OSH4 cells showed polarized localization of GFP-PHFAPP1 at 25 and 37°C, respectively. Scale bar, 5 μm. (B) Localization of GFP-PHFAPP1 and FM4-64 in osh1-7Δ/CEN osh4ts cells at 37°C. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. FM4-64 was added to cells and kept on ice to label the plasma membrane. Scale bar, 5 μm. (C) The PI4P probe GFP-2xPHOsh2 accumulates at polarized growth sites in sec6ts cells at the nonpermissive temperature. Wild-type and sec6ts cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 15 min. Cells shown are representative of >100 cells observed. Scale bar, 5 μm.
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Figure 1: Osh proteins regulate PI4P distribution in cells. (A) Localization of GFP-PHFAPP1 in osh1-7Δ /CEN OSH4 cells or osh1-7Δ /CEN osh4ts 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. Cells shown are representative of >100 cells observed. Note that 6 and 55% of osh1-7Δ/CEN osh4ts cells showed polarized localization of GFP-PHFAPP1 at 25 and 37°C, respectively, and 6 and 0% of osh1-7Δ/CEN OSH4 cells showed polarized localization of GFP-PHFAPP1 at 25 and 37°C, respectively. Scale bar, 5 μm. (B) Localization of GFP-PHFAPP1 and FM4-64 in osh1-7Δ/CEN osh4ts cells at 37°C. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 1 h. FM4-64 was added to cells and kept on ice to label the plasma membrane. Scale bar, 5 μm. (C) The PI4P probe GFP-2xPHOsh2 accumulates at polarized growth sites in sec6ts cells at the nonpermissive temperature. Wild-type and sec6ts cells were grown overnight at 25°C in a synthetic medium containing 2% glucose and then shifted to 37°C for 15 min. Cells shown are representative of >100 cells observed. Scale bar, 5 μm.
Mentions: The phosphoinositide PI4P is critical for the localization of a number of proteins involved in polarized secretion, including Sec2p (Mizuno-Yamasaki et al., 2010). Here we investigate how the level of PI4P is regulated on secretory vesicles. Studies from several groups have demonstrated that the oxysterol-binding proteins (Osh proteins) control the level and intracellular distribution of PI4P in the budding yeast Saccharomyces cerevisiae (LeBlanc and McMaster, 2010; Stefan et al., 2011). To begin our analysis of the role of Osh proteins in PI4P distribution, we used cells lacking all endogenous Osh proteins and solely expressing a temperature-sensitive osh4ts allele. Consistent with a prior report (Stefan et al., 2011), we observed that the PI4P probe GFP-PHFAPP1, consisting of the PH domain from the FAPP1 protein fused to green fluorescent protein (GFP), shifted from the Golgi apparatus to the plasma membrane in the osh1-7Δ osh4ts cells after a temperature shift to 37°C (Figure 1A). In addition, there was significantly more GFP-PHFAPP1 at polarized secretory sites, such as the tips of small buds and the necks of large-budded cells, in osh1-7Δ osh4ts cells at the nonpermissive temperature (Figure 1A). These GFP-PHFAPP1–positive structures could represent either secretory vesicles containing abnormally high levels of PI4P or an accumulation of PI4P produced at the plasma membrane. To differentiate these two possibilities, we labeled the plasma membrane with the lipophilic dye FM4-64. We noticed that the GFP-PHFAPP1 positive structures accumulated in the small buds of osh1-7Δ osh4ts cells were clearly internal to the FM4-64–labeled plasma membrane (Figure 1B). These results suggest that one or more Osh proteins are needed to reduce the level of PI4P on secretory vesicles.

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