Aitchison J D, Murray W W, Rachubinski R A
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
J Biol Chem. 1991 Dec 5;266(34):23197-203.
The gene encoding Candida tropicalis peroxisomal trifunctional enzyme, hydratase-dehydrogenase-epimerase (HDE), was expressed in both Candida albicans and Saccharomyces cerevisiae. The cellular location of HDE was determined by subcellular fractionation followed by Western blot analysis of peroxisomal and cytosolic fractions using antiserum specific for HDE. HDE was found to be exclusively targeted to and imported into peroxisomes in both heterologous expression systems. Deletion and mutational analyses were used to determine the regions within HDE which are essential for its targeting to peroxisomes. Deletion of a carboxyl-terminal tripeptide Ala-Lys-Ile completely abolished targeting of HDE to peroxisomes, whereas large internal deletions of HDE (amino acids 38-353 or 395-731) had no effect on HDE targeting to peroxisomes in either yeast. This tripeptide is similar to, but distinct from, other tripeptide peroxisomal targeting sequences (PTSs) as identified in peroxisomal firefly luciferase and four mammalian peroxisomal proteins. Substitutions within the carboxyl-terminal tripeptide (Ala----Gly and Lys----Gln) supported targeting of HDE to peroxisomes of C. albicans but not of S. cerevisiae. This is the first detailed analysis of the peroxisomal targeting signal in a yeast peroxisomal protein.
热带假丝酵母过氧化物酶体三功能酶（水合酶 - 脱氢酶 - 表异构酶，HDE）的编码基因在白色念珠菌和酿酒酵母中均有表达。通过亚细胞分级分离，然后使用针对HDE的抗血清对过氧化物酶体和胞质部分进行蛋白质免疫印迹分析，来确定HDE的细胞定位。发现在这两种异源表达系统中，HDE都专门靶向并导入过氧化物酶体。使用缺失和突变分析来确定HDE中对于其靶向过氧化物酶体至关重要的区域。羧基末端三肽Ala - Lys - Ile的缺失完全消除了HDE对过氧化物酶体的靶向作用，而HDE的大片段内部缺失（氨基酸38 - 353或395 - 731）对两种酵母中HDE靶向过氧化物酶体均无影响。该三肽与在过氧化物酶体荧光素酶和四种哺乳动物过氧化物酶体蛋白中鉴定出的其他三肽过氧化物酶体靶向序列（PTSs）相似，但又有所不同。羧基末端三肽内的替换（Ala→Gly和Lys→Gln）支持HDE靶向白色念珠菌的过氧化物酶体，但不支持其靶向酿酒酵母的过氧化物酶体。这是对酵母过氧化物酶体蛋白中过氧化物酶体靶向信号的首次详细分析。
