Lucero H A, Chojnicki E W, Mandiyan S, Nelson H, Nelson N
Department of Physiology, Boston University School of Medicine, Massachusetts 02118, USA.
J Biol Chem. 1995 Apr 21;270(16):9178-84. doi: 10.1074/jbc.270.16.9178.
The cloning, expression, and biochemical characterization of an essential gene of Saccharomyces cerevisiae that encodes for a new member of the TBP1-like subfamily of putative ATPases are described. The protein is 72% identical at the amino acid level to subunit four (S4) of the human 26 S protease and 73% identical to Schizosaccharomyces pombe MTS2 gene product. The purified, recombinant protein, designated Yhs4p, has an estimated molecular mass of 49 kDa and exhibits a Mg(2+)-dependent ATPase activity with nucleotide specificity and Km for ATP similar to those exhibited by the human 26 S protease. The observed ATPase activity was reduced by 73% upon the introduction of point mutation K229Q in the "P-loop" domain of the ATP-binding site relative to the nonmutated form of the protein. This is the first direct biochemical evidence supporting the putative ATPase activity of a member of the TBP1-like subfamily. Furthermore, the experimental results demonstrate a regulatory function for the amino-terminal region of the molecule. The amino-terminal truncated form of Yhs4p lacking two clusters of positively charged amino acids exhibits a greater ATPase activity. The ATPase activity of both the truncated and complete forms of Yhs4p is stimulated by polyanions. Polylysine partially inhibits the ATPase activity of the amino-terminal truncated form having no observable effect on the complete protein. N-Ethylmaleimide inhibits the ATPase activity of both forms of Yhs4p. We propose that Yhs4p ATPase may play an essential role in the regulatory function of the proteolytic activity of the yeast 26 S protease.
本文描述了酿酒酵母一个必需基因的克隆、表达及生化特性，该基因编码一种假定ATP酶的TBP1样亚家族新成员。该蛋白在氨基酸水平上与人类26S蛋白酶的亚基四（S4）有72%的同一性，与粟酒裂殖酵母MTS2基因产物有73%的同一性。纯化的重组蛋白命名为Yhs4p，估计分子量为49 kDa，表现出Mg(2+)依赖性ATP酶活性，其核苷酸特异性和对ATP的Km值与人26S蛋白酶相似。相对于未突变形式的蛋白，在ATP结合位点的“P环”结构域引入点突变K229Q后，观察到的ATP酶活性降低了73%。这是支持TBP1样亚家族成员假定ATP酶活性的首个直接生化证据。此外，实验结果证明了该分子氨基末端区域的调节功能。缺少两簇带正电荷氨基酸的Yhs4p氨基末端截短形式表现出更高的ATP酶活性。Yhs4p截短形式和完整形式的ATP酶活性均受到聚阴离子的刺激。聚赖氨酸部分抑制氨基末端截短形式的ATP酶活性，对完整蛋白没有明显影响。N-乙基马来酰亚胺抑制Yhs4p两种形式的ATP酶活性。我们认为Yhs4p ATP酶可能在酵母26S蛋白酶蛋白水解活性的调节功能中起重要作用。
