Makino Y, Yamano K, Kanemaki M, Morikawa K, Kishimoto T, Shimbara N, Tanaka K, Tamura T
Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-263, Japan.
J Biol Chem. 1997 Sep 12;272(37):23201-5. doi: 10.1074/jbc.272.37.23201.
SUG1 is an integral component of the 26 S proteasome. Belonging to a novel putative ATPase family, it shares four conserved motifs characteristic of ATP-dependent DNA/RNA helicases. Recombinant rat SUG1 (rSUG1) produced in Escherichia coli was highly purified and characterized in terms of its biochemical properties. The rSUG1 exhibited a Mg2+-dependent ATPase activity. The Km for ATP and Vmax of rSUG1 were 35 microM and 7 pmol of ATP/min/microg of protein, respectively. Both ATPase activity to release [32P]monophosphate and [32P]ATP-labeling activity were coordinately affected by cold ATP severely, GTP and UTP moderately, and CTP little. Interestingly, the rSUG1 ATPase activity was stimulated by poly(U) and poly(C), but not by poly(A), poly(G), or by any forms of DNAs tested. A UV cross-linking assay also indicated poly(U)- and poly(C)-stimulated labeling of rSUG1 with [alpha-32P]ATP. Moreover, the ATPase activity was facilitated by cellular poly(A)+ RNA, but not by poly(A)- RNA. RNA transcribed in vitro from cDNA encoding a b-Zip protein could stimulate the ATPase activity. This is the first report to demonstrate a specific RNA requirement for ATPase with respect to the proteasomal ATPases. Our present work suggests that SUG1 can specifically interact with protein-coding RNA (mRNA) and play some roles in mRNA metabolism.
SUG1是26S蛋白酶体的一个组成成分。它属于一个新的假定ATP酶家族，具有依赖ATP的DNA/RNA解旋酶特有的四个保守基序。对在大肠杆菌中产生的重组大鼠SUG1（rSUG1）进行了高度纯化，并对其生化特性进行了表征。rSUG1表现出Mg2+依赖的ATP酶活性。rSUG1对ATP的Km值和Vmax分别为35微摩尔和7皮摩尔ATP/分钟/微克蛋白质。释放[32P]单磷酸的ATP酶活性和[32P]ATP标记活性都受到冷ATP的严重协同影响，GTP和UTP的影响适中，CTP的影响很小。有趣的是，rSUG1的ATP酶活性受到聚（U）和聚（C）的刺激，但不受聚（A）、聚（G）或任何测试的DNA形式的刺激。紫外线交联试验也表明聚（U）和聚（C）刺激了rSUG1与[α-32P]ATP的标记。此外，细胞中的聚（A）+RNA促进了ATP酶活性，但聚（A）-RNA则没有。从编码b-Zip蛋白的cDNA体外转录的RNA可以刺激ATP酶活性。这是第一份证明蛋白酶体ATP酶对ATP酶有特定RNA需求的报告。我们目前的工作表明，SUG1可以与蛋白质编码RNA（mRNA）特异性相互作用，并在mRNA代谢中发挥一些作用。
