van der Leij F R, Kram A M, Bartelds B, Roelofsen H, Smid G B, Takens J, Zammit V A, Kuipers J R
Department of Pediatrics, Research Laboratory CMCV 2nd floor, Groningen University Hospital, NL-9700 RB Groningen, The Netherlands.
Biochem J. 1999 Aug 1;341 ( Pt 3)(Pt 3):777-84. doi: 10.1042/0264-6021:3410777.
Carnitine palmitoyltransferase I (CPT I) is a key enzyme in the regulation of beta-oxidation. The topology of this enzyme has been difficult to elucidate by biochemical methods. We studied the topology of a fusion protein of muscle-type CPT I (M-CPT I) and green fluorescent protein (GFP) by microscopical means. To validate the use of the fusion protein, designated CPT I-GFP, we checked whether the main characteristics of native CPT I were retained. CPT I-GFP was expressed in HeLa cells after stable transfection. Confocal laser scanning microscopy in living cells revealed an extranuclear punctate distribution of CPT I-GFP, which coincided with a mitochondrial fluorescent marker. Immunogold electron microscopy localized CPT I-GFP almost exclusively to the mitochondrial periphery and showed that the C-terminus of CPT I must be on the cytosolic face of the mitochondrial outer membrane. Western analysis showed a protein that was 6 kDa smaller than predicted, which is consistent with previous results for the native M-CPT I. Mitochondria from CPT I-GFP-expressing cells showed an increased CPT activity that was inhibited by malonyl-CoA and was lost on solubilization with Triton X-100. We conclude that CPT I-GFP adopts the same topology as native CPT I and that its C-terminus is located on the cytosolic face of the mitochondrial outer membrane. The evidence supports a recently proposed model for the domain structure of CPT I based on biochemical evidence.
肉碱棕榈酰转移酶I（CPT I）是调节β-氧化的关键酶。该酶的拓扑结构难以通过生化方法阐明。我们通过显微镜方法研究了肌肉型CPT I（M-CPT I）与绿色荧光蛋白（GFP）融合蛋白的拓扑结构。为验证名为CPT I-GFP的融合蛋白的用途，我们检查了天然CPT I的主要特性是否得以保留。稳定转染后，CPT I-GFP在HeLa细胞中表达。活细胞中的共聚焦激光扫描显微镜显示CPT I-GFP呈核外点状分布，这与线粒体荧光标记物相符。免疫金电子显微镜几乎将CPT I-GFP仅定位在线粒体周边，并表明CPT I的C末端必定位于线粒体外膜的胞质面上。蛋白质印迹分析显示有一种比预测小6 kDa的蛋白质，这与天然M-CPT I的先前结果一致。来自表达CPT I-GFP细胞的线粒体显示CPT活性增加，该活性受到丙二酰辅酶A的抑制，并且在用Triton X-100溶解后丧失。我们得出结论，CPT I-GFP采用与天然CPT I相同的拓扑结构，并且其C末端位于线粒体外膜的胞质面上。该证据支持了最近基于生化证据提出的CPT I结构域结构模型。
