Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM.
Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM.
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DOI：
10.1111/j.1600-0854.2011.01234.x
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发表时间：
2011-10
期刊：
影响因子：
--
通讯作者：
Menon AK
中科院分区：
文献类型：
--
作者：
Georgiev AG;Sullivan DP;Kersting MC;Dittman JS;Beh CT;Menon AK
内质网（ER）和质膜（PM）之间的固醇转运通过ATP依赖性的非囊泡机制发生，推测需要固醇转运蛋白（STP）。在酿酒酵母中，哺乳动物氧固醇结合蛋白（Osh 1 -7）的同源物已被提出作为STPs。为了评估这一建议，我们采取了两种方法。首先，我们使用脱氢麦角甾醇（DHE），通过荧光显微镜观察活细胞中甾醇的运动。DHE在缺氧条件下被引入PM中，并观察到在有氧生长细胞时重新分布为脂滴。再分配需要ATP和甾醇酰基转移酶Are 2，但不需要PM衍生的运输囊泡。DHE再分布在37°C下缺乏所有功能性奥什蛋白的条件酵母突变体（奥什Δ osh 4 - 1 ts）中强烈发生。在第二种方法中，我们使用脉冲追踪方案来分析代谢放射性标记的麦角固醇从ER到PM的运动。通过用甲基-β-环糊精从完整细胞中提取甾醇，在分离的PM富集级分中评估放射性标记的麦角甾醇到达PM。这些实验表明，尽管麦角固醇在Osh缺陷细胞中有效地从ER转运到PM，但其在PM内移动以与甲基-β-环糊精可提取的固醇池平衡的速率减慢。我们得出结论：（一）奥什蛋白在PM，ER和脂滴之间的非囊泡固醇运输的作用是最小的，或由其他机制和（ii），奥什蛋白调节组织的固醇在PM。
Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologues of the mammalian oxysterol-binding protein (Osh1–7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1ts) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in nonvesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.
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DOI：
10.1083/jcb.200806035
发表时间：
2008-12-15
期刊：
The Journal of cell biology
影响因子：
--
作者：
Grossmann G;Malinsky J;Stahlschmidt W;Loibl M;Weig-Meckl I;Frommer WB;Opekarová M;Tanner W
通讯作者：
Tanner W
影响因子：
6.8
作者：
Eliezer, David
通讯作者：
Eliezer, David
影响因子：
4
作者：
Beh, CT;Rine, J
通讯作者：
Rine, J
影响因子：
2.4
作者：
GREENBERG, ML;AXELROD, D
通讯作者：
AXELROD, D
影响因子：
3.9
作者：
KEESLER, GA;CASEY, WM;PARKS, LW
通讯作者：
PARKS, LW