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Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae.

Mioka T, Fujimura-Kamada K, Tanaka K - Microbiologyopen (2014)

Bottom Line: Furthermore, no reduction in the PS level was observed in a mutant with multiple flippase mutations.Because PS was not exposed in a mutant that accumulates ER or cis/medial-Golgi membranes, Golgi maturation seems to be a prerequisite for PS translocation.Our results suggest that an unknown mechanism, possibly a protein with flippase-like activity, acts in conjunction with known flippases to regulate PS translocation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, N15 W7, Kita-ku, Sapporo, 060-0815, Japan.

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Related in: MedlinePlus

Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in Neo1p-depleted cells. (A) Localization of GFP-Snc1p-pm with mRFP-Lact-C2 or Sec7p-mRFP in Neo1p-depleted cells. To deplete Neo1p, cells were incubated at 30°C for 10.5 h in SD-Ura (for Sec7p-mRFP) or SD-Leu (for mRFP-Lact-C2) medium. The strains used were PGAL1-3HA-NEO1 SEC7-mRFP1 (YKT1861) carrying pRS416-GFP-SNC1 pm (pKT1444) and PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1851) carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μm. (B) Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in the Neo1p-depleted sec6-4 mutant. Cells were incubated in SD-Leu medium at 30°C for 9.5 h to deplete Neo1p, followed by a shift to 30°C or 37°C for 1 h. The strain used was sec6-4 PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1852) carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μm. (C) GFP-Snc1p-pm, but not mRFP-Lact-C2, localized to ER-like membranes in Neo1p-depleted cells. Cells of PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1851) carrying pRS416-GFP-SNC1 pm (pKT1444) were grown as in (A). Arrows indicate the ER-like membrane structures. Bar, 5 μm.
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fig04: Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in Neo1p-depleted cells. (A) Localization of GFP-Snc1p-pm with mRFP-Lact-C2 or Sec7p-mRFP in Neo1p-depleted cells. To deplete Neo1p, cells were incubated at 30°C for 10.5 h in SD-Ura (for Sec7p-mRFP) or SD-Leu (for mRFP-Lact-C2) medium. The strains used were PGAL1-3HA-NEO1 SEC7-mRFP1 (YKT1861) carrying pRS416-GFP-SNC1 pm (pKT1444) and PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1851) carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μm. (B) Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in the Neo1p-depleted sec6-4 mutant. Cells were incubated in SD-Leu medium at 30°C for 9.5 h to deplete Neo1p, followed by a shift to 30°C or 37°C for 1 h. The strain used was sec6-4 PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1852) carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μm. (C) GFP-Snc1p-pm, but not mRFP-Lact-C2, localized to ER-like membranes in Neo1p-depleted cells. Cells of PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1851) carrying pRS416-GFP-SNC1 pm (pKT1444) were grown as in (A). Arrows indicate the ER-like membrane structures. Bar, 5 μm.

Mentions: We next examined the effect of Neo1p depletion on PS flipping. Because NEO1 is an essential gene (Hua et al. 2002), we depleted Neo1p using the glucose-repressible GAL1 promoter. When Neo1p was depleted for 10.5 h, GFP-Snc1p-pm accumulated at high levels in intracellular punctate structures (100%, n = 113 cells) that significantly overlapped with Sec7p-mRFP (70.8%, n = 144 structures) (Fig.4A, arrowheads). These punctate GFP-Snc1p-pm structures colocalized with mRFP-Lact-C2 (90.2%, n = 133 structures). Thus, Neo1p may be required for vesicle transport from the TGN, but PS was still flipped at the TGN in the absence of Neo1p. We then reasoned that if Neo1p depletion inhibits vesicle formation from the TGN, it should affect accumulation of SV-associated GFP-Snc1p-pm to the bud in the sec6-4 mutant. To test this idea, we constructed the PGAL1-NEO1 sec6-4 strain. Cells were grown at 30°C for 9.5 h in glucose-containing medium to deplete Neo1p, and then shifted to 37°C for 1 h. At 30°C, the Neo1p-depleted sec6-4 cells accumulated GFP-Snc1p-pm structures (100%, n = 104 cells) that colocalized with mRFP-Lact-C2 (86.3%, n = 139 punctate structures) (Fig4B), similar to the Neo1p-depleted cells described above. This localization pattern did not significantly change after the shift to 37°C: the GFP-Snc1p-pm structures (99.1%, n = 113 cells) were randomly localized, rather than restricted to the bud, and they colocalized with mRFP-Lact-C2 (88.9%, n = 144 punctate structures), suggesting that Neo1p depletion may affect SV formation.


Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae.

Mioka T, Fujimura-Kamada K, Tanaka K - Microbiologyopen (2014)

Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in Neo1p-depleted cells. (A) Localization of GFP-Snc1p-pm with mRFP-Lact-C2 or Sec7p-mRFP in Neo1p-depleted cells. To deplete Neo1p, cells were incubated at 30°C for 10.5 h in SD-Ura (for Sec7p-mRFP) or SD-Leu (for mRFP-Lact-C2) medium. The strains used were PGAL1-3HA-NEO1 SEC7-mRFP1 (YKT1861) carrying pRS416-GFP-SNC1 pm (pKT1444) and PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1851) carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μm. (B) Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in the Neo1p-depleted sec6-4 mutant. Cells were incubated in SD-Leu medium at 30°C for 9.5 h to deplete Neo1p, followed by a shift to 30°C or 37°C for 1 h. The strain used was sec6-4 PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1852) carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μm. (C) GFP-Snc1p-pm, but not mRFP-Lact-C2, localized to ER-like membranes in Neo1p-depleted cells. Cells of PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1851) carrying pRS416-GFP-SNC1 pm (pKT1444) were grown as in (A). Arrows indicate the ER-like membrane structures. Bar, 5 μm.
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fig04: Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in Neo1p-depleted cells. (A) Localization of GFP-Snc1p-pm with mRFP-Lact-C2 or Sec7p-mRFP in Neo1p-depleted cells. To deplete Neo1p, cells were incubated at 30°C for 10.5 h in SD-Ura (for Sec7p-mRFP) or SD-Leu (for mRFP-Lact-C2) medium. The strains used were PGAL1-3HA-NEO1 SEC7-mRFP1 (YKT1861) carrying pRS416-GFP-SNC1 pm (pKT1444) and PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1851) carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μm. (B) Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in the Neo1p-depleted sec6-4 mutant. Cells were incubated in SD-Leu medium at 30°C for 9.5 h to deplete Neo1p, followed by a shift to 30°C or 37°C for 1 h. The strain used was sec6-4 PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1852) carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μm. (C) GFP-Snc1p-pm, but not mRFP-Lact-C2, localized to ER-like membranes in Neo1p-depleted cells. Cells of PGAL1-3HA-NEO1 mRFP1-Lact-C2 (YKT1851) carrying pRS416-GFP-SNC1 pm (pKT1444) were grown as in (A). Arrows indicate the ER-like membrane structures. Bar, 5 μm.
Mentions: We next examined the effect of Neo1p depletion on PS flipping. Because NEO1 is an essential gene (Hua et al. 2002), we depleted Neo1p using the glucose-repressible GAL1 promoter. When Neo1p was depleted for 10.5 h, GFP-Snc1p-pm accumulated at high levels in intracellular punctate structures (100%, n = 113 cells) that significantly overlapped with Sec7p-mRFP (70.8%, n = 144 structures) (Fig.4A, arrowheads). These punctate GFP-Snc1p-pm structures colocalized with mRFP-Lact-C2 (90.2%, n = 133 structures). Thus, Neo1p may be required for vesicle transport from the TGN, but PS was still flipped at the TGN in the absence of Neo1p. We then reasoned that if Neo1p depletion inhibits vesicle formation from the TGN, it should affect accumulation of SV-associated GFP-Snc1p-pm to the bud in the sec6-4 mutant. To test this idea, we constructed the PGAL1-NEO1 sec6-4 strain. Cells were grown at 30°C for 9.5 h in glucose-containing medium to deplete Neo1p, and then shifted to 37°C for 1 h. At 30°C, the Neo1p-depleted sec6-4 cells accumulated GFP-Snc1p-pm structures (100%, n = 104 cells) that colocalized with mRFP-Lact-C2 (86.3%, n = 139 punctate structures) (Fig4B), similar to the Neo1p-depleted cells described above. This localization pattern did not significantly change after the shift to 37°C: the GFP-Snc1p-pm structures (99.1%, n = 113 cells) were randomly localized, rather than restricted to the bud, and they colocalized with mRFP-Lact-C2 (88.9%, n = 144 punctate structures), suggesting that Neo1p depletion may affect SV formation.

Bottom Line: Furthermore, no reduction in the PS level was observed in a mutant with multiple flippase mutations.Because PS was not exposed in a mutant that accumulates ER or cis/medial-Golgi membranes, Golgi maturation seems to be a prerequisite for PS translocation.Our results suggest that an unknown mechanism, possibly a protein with flippase-like activity, acts in conjunction with known flippases to regulate PS translocation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, N15 W7, Kita-ku, Sapporo, 060-0815, Japan.

Show MeSH
Related in: MedlinePlus