Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking.
Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking.
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DOI：
10.1073/pnas.2204396119
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发表时间：
2022-09-27
影响因子：
11.1
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中科院分区：
文献类型：
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鞘脂和胆固醇是膜的重要成分。很少有人知道它们的代谢是如何交叉调节的，以及这如何影响控制脂质运输的膜接触位点（MCS）。在这里，我们报告删除鞘氨醇激酶（SphKs），减少S1 P伴随增加其前体鞘氨醇和神经酰胺，减少内质网（ER）接触晚期内吞细胞器导致游离胆固醇的积累。然而，令人惊讶的是，胆固醇向ER的转运没有减少，因为SphKs的缺失促进了胆固醇转运蛋白Aster-B向质膜（PM）的募集，以促进胆固醇从PM向ER的转运。因此，SphKs和鞘脂代谢产物与ER一起管理不同的MCS，以控制胆固醇在不同细胞膜之间的运动。膜接触位点（MCS），细胞器之间的紧密膜并置，是脂质（包括胆固醇）在细胞器间转移、调节脂质稳态和协调内吞运输的平台。鞘氨醇激酶（SphKs）是将鞘氨醇磷酸化为具有生物活性的鞘氨醇-1-磷酸（S1 P）的两种同工酶，其与内吞运输有关。然而，SphK在调节膜动力学、脂质运输和MCS方面的生理功能尚不清楚。在这里，我们报告说，SphKs的删除减少S1 P伴随着其前体鞘氨醇和神经酰胺的增加，并显着减少内质网（ER）与晚期内吞细胞器的接触。表达的酶活性SphK 1，但不是催化失活，挽救了这些MCS的赤字。尽管游离胆固醇在SphK缺失细胞中的晚期内吞细胞器中积累，然而令人惊讶的是，胆固醇向ER的转运没有减少。重要的是，SphKs的缺失促进了ER驻留的胆固醇转移蛋白Aster-B（也称为GRAMD 1B）向质膜（PM）的募集，这与PM处更高的可及胆固醇和神经酰胺一致，以促进胆固醇从PM转移到ER。此外，神经酰胺增强体外结合的Aster-B β结构域的磷脂酰丝氨酸和胆固醇脂质体。我们的研究揭示了SphKs和鞘脂代谢物在ER网络和晚期内吞细胞器与PM之间控制不同细胞膜之间胆固醇运动的不同MCS中的先前未知作用。
Sphingolipids and cholesterol are important constituents of membranes. Very little is known how their metabolism is cross-regulated and how this affects membrane contact sites (MCS) that control lipid traffic. Here, we report deletion of sphingosine kinases (SphKs) that decreases S1P with concomitant increases in its precursors sphingosine and ceramide, reduced endoplasmic reticulum (ER) contacts with late endocytic organelles leading to accumulation of free cholesterol there. Surprisingly however, cholesterol transport to the ER was not reduced as deletion of SphKs promoted recruitment of cholesterol transfer protein Aster-B to the plasma membrane (PM) to facilitate transfer of cholesterol from the PM to the ER. Thus, SphKs and sphingolipid metabolites govern diverse MCS with the ER to control the movement of cholesterol between distinct cell membranes. Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.