PDF(Pubmed)
Abstract：
In budding yeast cells, much of the inner surface of the plasma membrane (PM) is covered with the endoplasmic reticulum (ER). This association is mediated by seven ER membrane proteins that confer cortical ER-PM association at membrane contact sites (MCSs). Several of these membrane \"tether\" proteins are known to physically interact with the phosphoinositide phosphatase Sac1p. However, it is unclear how or if these interactions are necessary for their interdependent functions. We find that SAC1 inactivation in cells lacking the homologous synaptojanin-like genes INP52 and INP53 results in a significant increase in cortical ER-PM MCSs. We show in sac1Δ, sac1tsinp52Δ inp53Δ, or Δ-super-tether (Δ-s-tether) cells lacking all seven ER-PM tethering genes that phospholipid biosynthesis is disrupted and phosphoinositide distribution is altered. Furthermore, SAC1 deletion in Δ-s-tether cells results in lethality, indicating a functional overlap between SAC1 and ER-PM tethering genes. Transcriptomic profiling indicates that SAC1 inactivation in either Δ-s-tether or inp52Δ inp53Δ cells induces an ER membrane stress response and elicits phosphoinositide-dependent changes in expression of autophagy genes. In addition, by isolating high-copy suppressors that rescue sac1Δ Δ-s-tether lethality, we find that key phospholipid biosynthesis genes bypass the overlapping function of SAC1 and ER-PM tethers and that overexpression of the phosphatidylserine/phosphatidylinositol-4-phosphate transfer protein Osh6 also provides limited suppression. Combined with lipidomic analysis and determinations of intracellular phospholipid distributions, these results suggest that Sac1p and ER phospholipid flux controls lipid distribution to drive Osh6p-dependent phosphatidylserine/phosphatidylinositol-4-phosphate counter-exchange at ER-PM MCSs.
摘要：
在出芽的酵母细胞中，质膜（PM）的大部分内表面被内质网（ER）覆盖。这种关联是由7种ER膜蛋白介导的，这些蛋白在膜接触位点（MCSs）赋予皮质ER-PM关联。已知这些膜“系链”蛋白中的几种与磷酸肌醇磷酸酶Sac1p物理相互作用。然而,尚不清楚这些相互作用对于它们相互依赖的功能是如何或是否必要的。我们发现，缺乏同源突触样基因INP52和INP53的细胞中的SAC1失活导致皮质ER-PMMCSs的显着增加。我们以sac1Δ显示，sac1tsinp52Δinp53Δ，或Δ-超级系链（Δ-s系链）细胞缺乏所有七个ER-PM系链基因，磷脂生物合成被破坏，磷酸肌醇分布被改变。此外，Δ-s系链细胞中的SAC1缺失导致致死性，表明SAC1和ER-PM系链基因之间存在功能重叠。转录组学分析表明，Δ-s系链或inp52Δinp53Δ细胞中的SAC1失活诱导ER膜应激反应，并引起自噬基因表达的磷酸肌醇依赖性变化。此外,通过分离拯救sac1ΔΔ-s系链致死性的高拷贝抑制剂，我们发现关键的磷脂生物合成基因绕过了SAC1和ER-PM系链的重叠功能,磷脂酰丝氨酸/磷脂酰肌醇-4-磷酸转移蛋白Osh6的过表达也提供了有限的抑制.结合脂质组学分析和细胞内磷脂分布的测定，这些结果表明Sac1p和ER磷脂通量控制脂质分布以驱动ER-PMMCSs上Osh6p依赖性磷脂酰丝氨酸/磷脂酰肌醇-4-磷酸的反交换。
