King S R, Ronen-Fuhrmann T, Timberg R, Clark B J, Orly J, Stocco D M
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Endocrinology. 1995 Nov;136(11):5165-76. doi: 10.1210/endo.136.11.7588255.
We have previously demonstrated that steroidogenic acute regulatory protein (StAR) is essential for the rate-limiting step in the acute regulation of steroidogenesis, which is the transport of cholesterol from the outer to the inner mitochondrial membrane. We have hypothesized that this transport occurs as the 37-kilodalton (kDa) precursor form of StAR is imported into the mitochondria and processed to its 30-kDa mature forms. Using an in vitro transcription and translation system in the presence of mitochondria isolated from unstimulated mouse MA-10 Leydig tumor cells, we now directly show that the 37-kDa form is indeed the cytosolic precursor of StAR and can be processed by mitochondria to all four 30-kDa mature forms. To determine the subcellular location of StAR in steroidogenic cells, ultrastructural immunocytochemistry was performed in adrenal zona fasciculata cells using the protein A-gold technique. We show that StAR is associated exclusively with the mitochondria. There, StAR is primarily localized in the intermembrane space and the intermembrane space side of the cristae membrane. StAR was shown to induce steroid production in isolated mitochondria. StAR protein was expressed in COS1 cells and the cell lysate, which was shown to contain abundant levels of StAR by Western blot analysis, was incubated with mitochondria isolated from unstimulated MA-10 cells. In these experiments, StAR increased steroid production by at least 4-fold over control mock-transfected lysate, and this increase was time and dose dependent. Furthermore, the increase in steroid production induced by StAR-containing lysate was not observed when COS1 lysate containing high levels of another mitochondrially imported protein, adrenodoxin, was used. We conclude from these results that in response to tropic hormone stimulation of steroidogenic cells, StAR is synthesized as a 37-kDa precursor, imported into the mitochondria, processed to its 30-kDa mature forms, and localized to the intermembrane space. During import and processing in vitro, StAR induces steroid production in isolated mitochondria in a specific manner.
我们之前已经证明，类固醇生成急性调节蛋白（StAR）对于类固醇生成急性调节中的限速步骤至关重要，该步骤是胆固醇从线粒体外膜转运至内膜。我们推测这种转运发生在StAR的37千道尔顿（kDa）前体形式被导入线粒体并加工成其30-kDa成熟形式之时。利用在存在从未受刺激的小鼠MA-10 Leydig肿瘤细胞分离的线粒体的情况下的体外转录和翻译系统，我们现在直接表明37-kDa形式确实是StAR的胞质前体，并且可以被线粒体加工成所有四种30-kDa成熟形式。为了确定StAR在类固醇生成细胞中的亚细胞定位，使用蛋白A-金技术在肾上腺束状带细胞中进行了超微结构免疫细胞化学分析。我们表明StAR仅与线粒体相关。在那里，StAR主要定位于膜间隙和嵴膜的膜间隙侧。已证明StAR可诱导分离的线粒体中类固醇的产生。StAR蛋白在COS1细胞中表达，通过蛋白质印迹分析显示含有丰富水平StAR的细胞裂解物与从未受刺激的MA-细胞中分离的线粒体一起孵育。在这些实验中，与对照空载体转染的裂解物相比，StAR使类固醇产生增加至少4倍，并且这种增加是时间和剂量依赖性的。此外，当使用含有高水平另一种线粒体导入蛋白肾上腺皮质铁氧化还原蛋白的COS1裂解物时，未观察到含StAR的裂解物诱导的类固醇产生增加。从这些结果我们得出结论，响应于类固醇生成细胞的促性腺激素刺激，StAR作为37-kDa前体合成，被导入线粒体，加工成其30-kDa成熟形式，并定位于膜间隙。在体外导入和加工过程中，StAR以特定方式诱导分离的线粒体中类固醇的产生。
