Defective assembly of a hybrid vacuolar H(+)-ATPase containing the mouse testis-specific E1 isoform and yeast subunits.
Defective assembly of a hybrid vacuolar H(+)-ATPase containing the mouse testis-specific E1 isoform and yeast subunits.
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DOI：
10.1016/j.bbabio.2008.06.011
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发表时间：
2008-10
期刊：
影响因子：
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通讯作者：
Kazuhiro Hayashi;G. Sun-Wada;Y. Wada;M. Nakanishi‐Matsui;M. Futai
中科院分区：
文献类型：
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作者：
Kazuhiro Hayashi;G. Sun-Wada;Y. Wada;M. Nakanishi‐Matsui;M. Futai
哺乳动物液泡型质子泵atp酶（v - atp酶）是多种多样的多亚基质子泵。它们由膜vo1和催化v1部门形成，其亚基具有细胞特异性或普遍存在的同种异构体。对一种独特的v - atp酶进行生化研究是困难的，因为在同一细胞中存在不同的同种异构体。然而，可以使用由同种异构体和其他酵母亚基形成的杂交v - atp酶来研究小鼠同种异构体的特性。如前所述，小鼠亚基E异构体E1（睾丸特异性）或E2（普遍存在）可以与酵母的其他亚基形成活性V-ATPase，但E1/酵母杂交V-ATPase在37°C时质子运输缺陷（孙-和田，g - h）。， Imai-Senga， Y., Yamamoto， A., Murata， Y., Hirata， T., Wada， Y., and Futai， M., 2002， J. Biol。化学。277,18098-18105)。在这项研究中，我们分析了E1/酵母杂交V-ATPase的特性，以了解E亚基的作用。当液泡或细胞的孵育温度升高至30℃时，缺陷杂化atp酶的质子转运可以可逆地恢复。对应于杂交V-ATPase的可逆缺陷，vo亚基a表位在37℃暴露于相应的抗体，但在30℃时变得不可接近。然而，免疫化学显示，v1区在37°C时仍与voa相关。对照酵母V-ATPase在37℃时具有活性，抗体无法接近其表位。葡萄糖耗散可以使v_1与Voin酵母菌分离，但在酸性ph下对杂交种只有轻微的影响。E1的Lys26和Val83之间的结构域包含8个在E1和E2之间不保守的残基，这是杂交种独特性质的原因。这些结果表明，亚基E，特别是其氨基末端结构域，在v - atp酶亚基在液泡膜上的组装中起着相关的作用。
Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane Voand catalytic V1sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 °C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098–18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 °C. Corresponding to the reversible defect of the hybrid V-ATPase, the Vosubunit a epitope was exposed to the corresponding antibody at 37 °C, but became inaccessible at 30 °C. However, the V1sector was still associated with Voat 37 °C, as shown immunochemically. The control yeast V-ATPase was active at 37 °C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V1from Voin yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.