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体膜蛋白的结构和功能，并研究了其中一种蛋白在细胞内向过氧膜的转运。获得了以下新证据：1.为了鉴定新的过氧化酶体膜蛋白，我们选择了几种抗大鼠肝脏过氧化酶体膜的单抗。利用其中一种抗体，鉴定出一种新的57 kDa蛋白。结论：1.蛋白质暴露于过氧化体膜的胞液面，随着过氧化物酶的增殖，蛋白质的量增加。为了研究过氧化体膜蛋白的结构，用抗过氧化体膜蛋白抗体筛选了大鼠肝脏中的cDNA文库。选择阳性的cDNAs进行测序。其中一个被鉴定为一种新的37 kDa蛋白。其他三个克隆也被鉴定为同源蛋白，如HMG-CoA还原酶、亮氨酸氨基肽酶和H^+-A…的d亚基TP合酶活性分别较高。在大鼠肝癌H4-II-E细胞中研究了一种主要的过氧化体膜蛋白(69 KDa)的生物合成和细胞内转运。用蛋氨酸标记细胞并追逐不同时间。免疫沉淀法分析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，在15min内被转运到过氧体，并在没有蛋白降解处理的情况下整合到过氧体膜中。质子离子载体CCCP抑制蛋白质向过氧异构体膜的转运。具有两簇负电荷的聚磺酸盐化合物苏拉明被发现是一种有效的过氧化体蛋白进口抑制剂。苏拉明通过与过氧膜蛋白相互作用来抑制进口。苏拉明在进口过程中优先影响了过氧化体蛋白的易位步骤。这些结果表明，过氧酶体膜蛋白(S)在将过氧酶体蛋白输入过氧酶体中具有重要作用。较少

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