Structure and function of peroxisomal membrane proteins and assembly of the proteins into peroxisomal membranes

在这项研究中，我们研究了几种过氧化物酶体膜蛋白的结构和功能，并表征了其中一种蛋白到过氧化物酶体膜的细胞内转运。获得了以下新的证据：为了鉴定新的过氧化物酶体膜蛋白，选择了几种抗大鼠肝脏过氧化物酶体膜的单克隆抗体。利用其中一种抗体，鉴定出一种新的57 kDa蛋白。蛋白质暴露在过氧化物酶体膜的细胞质表面，蛋白质的数量与过氧化物酶体的增殖平行增加。为了检测过氧化物酶体膜蛋白的结构，利用抗过氧化物酶体膜蛋白抗体筛选了大鼠肝脏lambda gtll CDNA文库。选择阳性cdna并测序。其中一个被鉴定为一个新的37 kDa蛋白。另外3个克隆分别为HMG-COA还原酶、亮氨酸氨基肽酶和H^+-A…More TP合成酶的d亚基同源蛋白。研究了大鼠肝癌H4-II-E细胞中一种主要过氧化物酶体膜蛋白（69 kDa）的生物合成和细胞内转运。细胞用[^<35>S]蛋氨酸标记，并进行不同时间的追踪。免疫沉淀法分析了^<35>S-69 kDa蛋白的亚细胞分布。结果表明，新合成的69 kDa蛋白与细胞质中的一些大分子结合，然后在15分钟内被转运到过氧化物酶体，并在未经蛋白水解的情况下整合到过氧化物酶体膜中。质子离子载体CCCP抑制了蛋白质向过氧化物异构体膜的转运。具有两簇负电荷的聚磺酸化合物苏拉明被发现是一种有效的过氧化物蛋白进口抑制剂。苏拉明通过与过氧化物膜蛋白相互作用抑制进口。苏拉明优先影响进口过氧化物酶体蛋白的易位步骤。这些结果表明，过氧化物酶体膜蛋白在过氧化物酶体蛋白输入中起着重要的作用。少

In this study, we investigated structure and function of several peroxisomal membrane proteins and characterized intracellular transport of one of the proteins to peroximal membranes. Following new evidences were obtained.1. In order to identify novel peroxisomal membrane proteins, several monoclonal antibodies against rat liver peroxisomal membranes were selected. Using one of the antibodies, a novel 57 kDa protein was identified. The protein is exposed to the cytosolic face of the peroxisomal membrane and the amount of the protein increased in paralleled with proliferation of peroxisomes.2. In order to examine structure of peroxisomal membrane proteins, a rat liver CDNA library in lambda gtll was screened by anti-peroxisomal membrane proteins antibodies. Positive cDNAs were selected and sequenced. One of them was identified as a novel 37 kDa protein. Other three clones were also identified as homologous proteins such as HMG-COA reductase, leucine aminopeptidase and subunit d of H^+-A … More TP synthase respectively.3. Biosynthesis and intracellular transport of a major peroxisomal membrane protein (69 kDa) were investigated in rat hepatoma H4-II-E cells. The cells were labelled with [ ^<35>S] methionine and chased for various periods of time. Subcellular distribution of the ^<35>S-69 kDa protein was analyzed by immunoprecipitation. The results suggest that the newly synthesized 69 kDa protein associated with some macromolecules in the cytoplasm and then was transported to peroxisomes within 15 min, and integrated into peroxisomal membranes without proteolytic processing. Proton ionophore CCCP inhibited the transport of the protein to peroxoisomal membranes.4. Polysulfonate compound suramin which have two clusters of negative charges was founded to be a potent inhibitor of the import of peroxiomal proteins. Suramin inhibited the import by interacting with peroximal membrane proteins. Suramin affected preferentially translocation step of peroxisomal proteins in the import. These results suggest that peroxisomal membrane protein(s) has important role of import of peroxisomal proteins into peroxisomes. Less