The ORMs interact with transmembrane domain 1 of Lcb1 and regulate serine palmitoyltransferase oligomerization, activity and localization
The ORMs interact with transmembrane domain 1 of Lcb1 and regulate serine palmitoyltransferase oligomerization, activity and localization
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DOI：
10.1016/j.bbalip.2018.11.007
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发表时间：
2019-03-01
影响因子：
4.8
通讯作者：
Dunn, Teresa M.
中科院分区：
文献类型：
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作者：
Han, Gongshe;Gupta, Sita D.;Dunn, Teresa M.
丝氨酸棕榈酰基转移酶(SPT)是一种内质网定位的膜酶，由催化活性的LCB1/LCB2异源二聚体和一个小的激活亚基(酵母中为Tsc3，哺乳动物中为SSSPTS)组成，受进化上保守的Orms家族的负调控。在酵母中，SPT、Orms和PI4P磷酸酶SACL在“斑点”复合体中相互融合。然而，无论是ORM抑制SPT的机制还是ORM和SACL与SPT相互作用的细节都不清楚。在这里，我们报告了Lcb1的第一个跨膜结构域(TMD1)是ORM与SPT结合所必需的。结合的丧失不是由于Lcb1的膜拓扑改变所致，因为用异源TMD取代TMD1可以恢复膜拓扑，但不能恢复ORM结合。TMD1缺失也消除了SPT寡聚体对ORM的依赖形成，通过免疫共沉淀分析和体内成像进行了评估。缺乏去抑制磷酸化位点的ORM的表达会导致结构性的SPT寡聚，而仿磷ORM在任何条件下都不能诱导寡聚。值得注意的是，当LCB1-RFP和LCB1ATMD1-GFP共表达时，外周ER中有更多的LCB1ATIVID1-GFP，这表明ORM的调节部分是通过SPT的重新分布来完成的。Tsc3缺失并不取消ORM对SPT的抑制，表明ORM不仅仅是阻止Tsc3激活。SACL与SPT的结合需要Tsc3，但不需要ORM，SACL不影响ORM介导的SPT寡聚。最后，缺乏SPT ORM调控的酵母突变株需要LCB-P裂解酶Dpl1将长链碱基维持在亚致死水平。
Serine palmitoyltransferase (SPT), an endoplasmic reticulum-localized membrane enzymecomposed of acatalytic LCB1/LCB2 heterodimer and a small activating subunit (Tsc3 in yeast; ssSPTs in mammals), is negatively regulated by the evolutionarily conserved family of proteins known as the ORMs. In yeast, SPT, the ORMs, and the PI4P phosphatase Sacl, copurify in the "SPOTs" complex. However, neither the mechanism of ORM inhibition of SPT nor details of the interactions of the ORMs and Sacl with SPT are known. Here we report that the first transmembrane domain (TMD1) of Lcbl is required for ORM binding to SPT. Loss of binding is not due to altered membrane topology of Lcbl since replacing TMD1 with a heterologous TMD restores membrane topology but not ORM binding. TMD1 deletion also eliminates ORM-dependent formation of SPT oligomers as assessed by co-immunoprecipitation assays and in vivo imaging. Expression of ORMs lacking derepressive phosphorylation sites results in constitutive SPT oligomerization, while phosphomimetic ORMs fail to induce oligomerization under any conditions. Significantly, when LCB1-RFP and LCB1ATMD1-GFP were coexpressed, more LCB1ATIVID1-GFP was in the peripheral ER, suggesting ORM regulation is partially accomplished by SPT redistribution. Tsc3 deletion does not abolish ORM inhibition of SPT, indicating the ORMs do not simply prevent activation by Tsc3. Binding of Sacl to SPT requires Tsc3, but not the ORMs, and Sacl does not influence ORM-mediated oligomerization of SPT. Finally, yeast mutants lacking ORM regulation of SPT require the LCB-P lyase Dpl1 to maintain long-chain bases at sublethal levels.