The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase.
The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase.
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DOI：
10.1016/s0021-9258(19)50651-2
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发表时间：
1992-02
期刊：
影响因子：
--
通讯作者：
E. Olender;R. Simon
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文献类型：
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作者：
E. Olender;R. Simon
我们提出的证据表明，3-羟基-3-甲基戊二酰辅酶A还原酶的氨基末端39个残基区域，包括可能的第一跨膜跨度，是将HMG-CoA还原酶靶向内质网的信号序列。这一证据是基于对体外转录/易位系统的分级、内切糖苷酶-H敏感性和蛋白酶保护分析，该系统以HMG-CoA还原酶的突变cDNA为编程，该突变的cDNA被删除，编码除第一个跨膜跨膜之外的所有假定跨膜跨膜的序列。我们发现，只有在信号识别粒子存在的情况下，这个突变的cDNA的蛋白质产物才能与微生物体结合、糖基化或不被蛋白质降解。此外，我们还提出了一个由八个跨膜跨度组成的HMG-CoA还原酶拓扑模型的证据。这一证据是基于刀豆蛋白A结合试验，对融合蛋白HMGal(Skalnik，D.G.，Narita，H.，Kent，C.和Simoni，R.D.(1988)J.Biol)的一系列突变体中的每个突变的工程糖基化位点进行体内糖基化结合试验。化学。263、6836-6841)。这一系列突变体的设计是这样的，对于跨膜跨度之间的每个连接体片段，构建了一个突变体，在该连接体片段中引入了一个工程糖基化位点。我们发现，只有在跨膜跨度1和2、3和4以及5和6之间连接区段具有糖基化位点的突变体是糖基化的。这些结果支持HMG-CoA还原酶拓扑结构的八跨膜跨度模型，而与七跨膜跨度模型不一致。
We present evidence that the amino-terminal 39 residue region of 3-hydroxy-3-methylglutaryl- (HMG) CoA reductase, which includes the putative first transmembrane span, is a signal sequence for targeting HMG-CoA reductase to the endoplasmic reticulum. This evidence is based upon fractionation, endoglycosidase-H sensitivity and protease protection assays on an in vitro transcription/translocation system programmed with a mutant cDNA of HMG-CoA reductase that is deleted for sequences coding for all of the putative transmembrane spans except the first. We show that the protein product of this mutant cDNA is associated with microsomes, glycosylated, or protected from proteolysis only in the presence of Signal Recognition Particle. Also, we present evidence for a topological model of HMG-CoA reductase that consists of eight transmembrane spans. This evidence is based upon a concanavalin A binding assay for in vivo glycosylation of an engineered glycosylation site in each of a series of mutants of the fusion protein, HMGal (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). This series of mutants was designed such that for each linker segment between transmembrane spans, a mutant was constructed with an engineered glycosylation site introduced into that linker segment. We show that only the mutants with glycosylation sites in the linker segments between transmembrane spans 1 and 2, 3 and 4, and 5 and 6 are glycosylated. These results support an eight transmembrane span model for the topology of HMG-CoA reductase and are inconsistent with a seven-transmembrane span model.