Kiel J A, Hilbrands R E, van der Klei I J, Rasmussen S W, Salomons F A, van der Heide M, Faber K N, Cregg J M, Veenhuis M
Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Yeast. 1999 Aug;15(11):1059-78. doi: 10.1002/(SICI)1097-0061(199908)15:11<1059::AID-YEA434>3.0.CO;2-I.
We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome-deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Deltapex1 or Deltapex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co-sedimented with the peroxisomal membrane protein HpPex3p in both wild-type cells and in Deltapex4, Deltapex8 or Deltapex14 cells. Both proteins are loosely membrane-bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co-overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane-bound, but only partially co-localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity.
我们通过对相应的过氧化物酶体缺陷（pex）突变体进行功能互补，克隆了多形汉逊酵母的PEX1和PEX6基因。基因产物HpPex1p和HpPex6p是ATP酶，它们都属于AAA蛋白家族。缺失任一基因（Deltapex1或Deltapex6）的细胞的特征是存在小的过氧化物酶体残余物，其中含有过氧化物酶体膜蛋白和少量基质蛋白。然而，大部分基质蛋白存在于细胞质中。在细胞分级分离研究中，HpPex1p和HpPex6p在野生型细胞以及Deltapex4、Deltapex8或Deltapex14细胞中均与过氧化物酶体膜蛋白HpPex3p共同沉降。这两种蛋白都松散地结合在膜上，面向细胞质。此外，HpPex1p和HpPex6p在体内存在物理和功能上的相互作用。PEX6的过表达导致过氧化物酶体基质蛋白导入缺陷。相比之下，PEX1的过表达对细胞无害。有趣地是，HpPex1p的共同过量产生挽救了由HpPex6p过量产生导致的蛋白导入缺陷。过量产生的HpPex1p和HpPex6p主要仍结合在膜上，但仅部分与过氧化物酶体膜蛋白HpPex3p共定位。我们的数据表明，HpPex1p和HpPex6p在与过氧化物酶体膜相关的蛋白复合物中发挥作用，并且过量产生、定位错误的HpPex6p会阻止HpPex1p到达其活性位点。
