Shen Jian, Hsu Ching-Mei, Kang Bae-Kwang, Rosen Barry P, Bhattacharjee Hiranmoy
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.
Biometals. 2003 Sep;16(3):369-78. doi: 10.1023/a:1022504311669.
Homologues of the bacterial ArsA ATPase are found in nearly every organism. While the enzyme is involved in arsenic detoxification in bacteria, the roles of eukaryotic homologues have not been identified. This article reports the function of the Saccharomyces cerevisiae homologue encoded by ARR4 gene (YDL100c ORF). Disruption of ARR4 was not lethal, but the disrupted strain displayed increased sensitivity to As3+, As5+, Co2+, Cr3+, Cu2+ or VO4(3-) salts and temperature. A plasmid-encoded copy of a wild-type ARR4 gene could complement the heat- or metal-related stress responses. Mutation of a codon within the consensus sequence for the nucleotide-binding site resulted in loss of complementation of the disrupted strain and produced a dominant negative phenotype in a wild type strain. Wild type and mutant Arr4p were purified from Escherichia coli. The wild type protein exhibited a low level of ATPase activity, and the mutant was inactive. The purified ATPase eluted as a dimer of 80-kDa species. A fusion of ARR4 and the GFP (green fluorescent protein) gene was constructed. The gene fusion was able to complement stress-related phenotype of the ARR4 disruption. Under non-stress conditions, GFP fluorescence was found diffusely in the cytosol. Under stress conditions GFP was localized in a few punctate bodies resembling late endosomes. It is proposed that under heat or metal stress, the soluble ATPase becomes membrane-associated, perhaps through interaction with a partner protein, and that this complex is involved in stress tolerance.
几乎在每种生物体中都能找到细菌ArsA ATP酶的同源物。虽然该酶在细菌的砷解毒过程中发挥作用，但真核生物同源物的功能尚未明确。本文报道了由ARR4基因（YDL100c开放阅读框）编码的酿酒酵母同源物的功能。ARR4基因的破坏并非致命，但破坏后的菌株对As3 +、As5 +、Co2 +、Cr3 +、Cu2 +或VO4(3-)盐以及温度表现出更高的敏感性。野生型ARR4基因的质粒编码拷贝能够补充与热或金属相关的应激反应。核苷酸结合位点共有序列内一个密码子的突变导致破坏菌株的互补作用丧失，并在野生型菌株中产生显性负性表型。野生型和突变型Arr4p从大肠杆菌中纯化得到。野生型蛋白表现出低水平的ATP酶活性，而突变型则无活性。纯化的ATP酶以80 kDa的二聚体形式洗脱。构建了ARR4与绿色荧光蛋白（GFP）基因的融合体。该基因融合体能够补充ARR4破坏相关的应激表型。在非应激条件下，GFP荧光在细胞质中呈弥散分布。在应激条件下，GFP定位于一些类似晚期内体的点状小体中。有人提出，在热或金属应激下，可溶性ATP酶可能通过与伴侣蛋白相互作用而与膜结合，并且这种复合物参与应激耐受性。
