Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities
Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities
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DOI：
10.1073/pnas.0811269106
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发表时间：
2009-05-19
影响因子：
11.1
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.
丝氨酸棕榈酰转移酶（SPT）催化鞘脂生物合成的第一个关键步骤。在酵母中，SPT由2个高度相关的亚基Lcb1p和Lcb2p以及第三个亚基Tsc3p的异二聚体组成，Tsc3p显著增加酶活性，并且是在高温下生长所必需的。已经鉴定了Lcb1p和Lcb2p的高等真核直向同源物，但是SPT活性与这些亚基的共表达并不高度相关，并且还没有鉴定出Tsc3p的直向同源物。在这里，我们报告发现的2种蛋白质，ssSPTa和ssSPTb，尽管共享没有同源性Tsc3p，每个大大提高了活性的哺乳动物SPT表达在酵母或哺乳动物细胞，因此定义了一个进化保守的低分子量蛋白质家族，赋予充分的酶活性。这2种ssSPT亚型共有一个保守的疏水性中心结构域，预测该结构域位于膜中，并且通过正分裂泛素双杂交分析评估，每种亚型均与hLCB 1和hLCB 2相互作用。这些小亚基的存在，沿着2 hLCB 2亚型，表明存在4种不同的人SPT同工酶。当每种SPT同工酶在缺乏内源性SPT活性的酵母或CHO LyB细胞中表达时，其体外酶活性的表征和长链碱基（LCB）分析揭示了酰基辅酶A偏好的差异，这为在哺乳动物细胞中观察到的LCB多样性提供了潜在的解释。
Serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid biosynthesis. In yeast, SPT is composed of a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly and is required for growth at elevated temperatures. Higher eukaryotic orthologs of Lcb1p and Lcb2p have been identified, but SPT activity is not highly correlated with coexpression of these subunits and no ortholog of Tsc3p has been identified. Here, we report the discovery of 2 proteins, ssSPTa and ssSPTb, which despite sharing no homology with Tsc3p, each substantially enhance the activity of mammalian SPT expressed in either yeast or mammalian cells and therefore define an evolutionarily conserved family of low molecular weight proteins that confer full enzyme activity. The 2 ssSPT isoforms share a conserved hydrophobic central domain predicted to reside in the membrane, and each interacts with both hLCB1 and hLCB2 as assessed by positive split ubiquitin 2-hybrid analysis. The presence of these small subunits, along with 2 hLCB2 isofoms, suggests that there are 4 distinct human SPT isozymes. When each SPT isozyme was expressed in either yeast or CHO LyB cells lacking endogenous SPT activity, characterization of their in vitro enzymatic activities, and long-chain base (LCB) profiling revealed differences in acyl-CoA preference that offer a potential explanation for the observed diversity of LCB seen in mammalian cells.