Identification of a yeast peroxisomal member of the family of AMP-binding proteins
Identification of a yeast peroxisomal member of the family of AMP-binding proteins
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DOI：
10.1111/j.1432-1033.1996.0468h.x
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发表时间：
1996-09-01
期刊：
影响因子：
--
通讯作者：
Erdmann, R
中科院分区：
文献类型：
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作者：
Blobel, F;Erdmann, R
我们建立了一个反向遗传学的方法来确定过氧化物酶体蛋白参与过氧化物酶体脂肪酸代谢的酿酒酵母。通过连续提取纯化的过氧化物酶体并通过HPLC和SDS/PAGE纯化来分离推定的过氧化物酶体外周膜蛋白。通过肽序列分析鉴定了6种蛋白质，包括酰基辅酶A氧化酶和过氧化物酶体β-氧化系统的三功能酶以及过氧化物酶体苹果酸脱氢酶3和肉毒碱乙酰转移酶。此外，两个以前未知的假定过氧化物酶体蛋白被确定，一个未知的40 kDa的蛋白质和蛋白质，我们命名为Pcs 60 p，两个分离的蛋白质馏分的主要成分。Pcs 60 p由S.酿酒酵母，由543个氨基酸组成，分子量为60.5 Ma。生化，免疫荧光显微镜和免疫细胞化学数据证实，Pcs 60 p是过氧化物酶体外周膜蛋白，但该蛋白质也定位于过氧化物酶体基质。与过氧化物酶体内定位一致，过氧化物酶体靶向信号1（PTS 1）的共有序列存在于Pcs 60 p的最末端C末端。缺失研究表明，过氧化物酶体定位的蛋白质依赖于这个信号序列的存在。Pcs 60 p的表达受油酸高度诱导，然而，该蛋白不适于以油酸作为单一碳源生长。Pcs 60 p属于通过AMP与其底物的ATP依赖性共价结合起作用的蛋白质家族，并且与大肠杆菌长链酰基辅酶A合成酶显示出最高程度的相似性。
We established a reverse-genetic approach to identify peroxisomal proteins involved in peroxisomal fatty acid metabolism of Saccharomyces cerevisiae. Putative peroxisomal peripheral membrane proteins were isolated by successive extraction of purified peroxisomes and purified by HPLC and SDS/PAGE. Six proteins were identified by peptide sequence analysis, including acyl-CoA oxidase and a trifunctional enzyme of the peroxisomal beta-oxidation system as well as peroxisomal malate dehydrogenase 3 and carnitine acetyltransferase. In addition two previously unknown putative peroxisomal proteins were identified, an unknown 40-kDa protein and a protein which we named Pcs60p, both major constituent of the isolated protein fraction. Pcs60p is encoded by ORF Z36091 of chromosome II from S. cerevisiae and consists of 543 amino acids with a molecular mass of 60.5 Ma. Biochemical, immunofluorescence microscopy and immunocytochemical data confirmed that Pcs60p is a peroxisomal peripheral membrane protein but the protein is also localized in the peroxisomal matrix. Consistent with the intraperoxisomal localization, the consensus sequence for a peroxisomal-targeting signal 1 (PTS1) is present at the extreme C-terminus of Pcs60p. Deletion studies revealed that the peroxisomal localization of the protein depends on the presence of this signal sequence. Expression oi Pcs60p is highly inducible by oleic acid, however, the protein is dispensable for growth on oleic acid as single carbon source. Pcs60p belongs to the family of proteins which act via an ATP-dependent covalent binding of AMP to their substrates and shows the highest degree of similarity to the Escherichia coli long chain acyl-CoA synthetase.