Yoshimura Shige H, Iwasaka Shizuka, Schwarz Wolfgang, Takeyasu Kunio
Graduate School of Biostudies, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
J Cell Sci. 2008 Jul 1;121(Pt 13):2159-68. doi: 10.1242/jcs.022905. Epub 2008 Jun 3.
The cell-surface expression and function of multisubunit plasma membrane proteins are regulated via interactions between catalytic subunits and auxiliary subunits. Subunit assembly in the endoplasmic reticulum is required for the cell-surface expression of the enzyme, but little is known about subunit interactions once it reaches the plasma membrane. Here we performed highly quantitative analyses of the catalytic (alpha1) and auxiliary (beta1 and beta3) subunits of Na(+)/K(+)-ATPase in the HeLa cell plasma membrane using isoform-specific antibodies and a cell-surface protein labeling procedure. Our results indicate that although the beta-subunit is required for the cell-surface expression of the alpha-subunit, the plasma membrane contains more alpha-subunits than beta-subunits. Pulse-labeling and chasing of the cell-surface proteins revealed that degradation of the beta-subunits was much faster than that of the alpha1-subunit. Ubiquitylation, as well as endocytosis, was involved in the fast degradation of the beta1-subunit. Double knockdown of the beta1- and beta3-subunits by RNAi resulted in the disappearance of these beta-subunits but not the alpha1-subunit in the plasma membrane. All these results indicate that the alpha- and beta-subunits of Na(+)/K(+)-ATPase are assembled in the endoplasmic reticulum, but are disassembled in the plasma membrane and undergo different degradation processes.
多亚基质膜蛋白的细胞表面表达和功能通过催化亚基与辅助亚基之间的相互作用来调节。内质网中的亚基组装是该酶细胞表面表达所必需的，但对于其到达质膜后的亚基相互作用却知之甚少。在此，我们使用亚型特异性抗体和细胞表面蛋白标记程序，对HeLa细胞质膜中Na(+)/K(+)-ATP酶的催化（α1）亚基和辅助（β1和β3）亚基进行了高度定量分析。我们的结果表明，尽管β亚基是α亚基细胞表面表达所必需的，但质膜中α亚基的含量比β亚基多。对细胞表面蛋白进行脉冲标记和追踪显示，β亚基的降解速度比α1亚基快得多。泛素化以及内吞作用参与了β1亚基的快速降解。通过RNAi对β1和β3亚基进行双重敲低导致这些β亚基在质膜中消失，但α1亚基并未消失。所有这些结果表明，Na(+)/K(+)-ATP酶的α和β亚基在内质网中组装，但在质膜中分解并经历不同的降解过程。
