Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Biológica, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Buenos Aires, Argentina.
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Biológica, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Buenos Aires, Argentina.
Biochem Biophys Res Commun. 2021 Jul 23;563:113-118. doi: 10.1016/j.bbrc.2021.05.054. Epub 2021 Jun 1.
The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the "arm-like" domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the "arm-like" domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the "arm-like" domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation.
酵母 Spf1p P5A-ATP 酶可将错误定位的蛋白质的跨膜肽从内质网主动转运。Spf1p 功能丧失会导致多效性内质网应激表型，与金属离子、脂质、蛋白质折叠、糖基化和膜插入的动态平衡改变有关。P5A-ATP 酶的一个独特特征是存在一个延伸的插入，称为“臂状”结构域，连接磷酸化结构域（P）与靠近肽基底物结合口袋的跨膜片段 M5。在这里，我们构建并表征了 Spf1p 的 Δarm 突变体，该突变体缺失了“臂状”结构域的 117 个氨基酸片段。Δarm 突变体能以野生型酶最大速率的 50%水解 ATP。用非核苷酸底物类似物 pNPP，突变体的水解活性下降到 10%。该突变体对 ATP 的表观亲和力与野生型相似。当与 ATP 孵育时，Δarm 突变体产生的催化磷酸酶水平较低，与 ATP 酶活性成比例。这些结果表明“臂状”结构域对于水解活性不是必需的，并表明它对于 Spf1p 处于磷酸化准备构象的稳定是必需的。
