ROMK1 channel activity is regulated by monoubiquitination.
ROMK1 channel activity is regulated by monoubiquitination.
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ROMK1 通道活性受单泛素化调节。
DOI：
10.1073/pnas.0409767102
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Wang,Wen-Hui
中科院分区：
文献类型：
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作者：
Lin,Dao-Hong;Sterling,Hyacinth;Wang,Zhijian;Babilonia,Elisa;Yang,Baofeng;Dong,Ke;Hebert,StevenC;Giebisch,Gerhard;Wang,Wen-Hui
蛋白质的泛素化可以发出其降解的信号，改变其活性或将其靶向至特定的膜或细胞器。在这里，我们表明，monoubiquitination调节质膜丰度和钾通道，ROMK的功能。从肾皮质和外髓质与ROMK抗体获得的蛋白质的免疫沉淀显示，该通道是monoubiquitinated。为了确定ROMK 1上的泛素结合位点，将ROMK 1的所有细胞内赖氨酸（Lys）残基单独突变为精氨酸（Arg），并使用双电极电压钳来测量ROMK 1通道活性。只有当Lys-22突变为Arg时，ROMK 1通道活性才从8.1 μ A增加到27.2 μA。Western blotting未能检测到注射R1 K22 R的卵母细胞中泛素化的ROMK 1。膜片钳实验表明，R1 K22 R的生物物理特性是相同的野生型ROMK 1。虽然GFP-ROMK 1和GFP-R1 K22 R在卵母细胞中的总蛋白表达水平相似，但共聚焦显微镜显示，注射GFP-R1 K22 R的卵母细胞的表面荧光强度高于GFP-ROMK 1。此外，HEK 293细胞中表达的ROMK 1和R1 K22 R蛋白的生物素标记显示Lys-22突变体通道的表面表达增加。最后，在COS 7细胞中表达R1 K22 R显著刺激ROMK 1的表面表达。我们的结论是，ROMK 1可以monoubiquitinated和赖氨酸-22是一个泛素结合位点。因此，ROMK 1的单泛素化通过减少通道蛋白的表面表达来调节通道活性。这一发现暗示了一个单一的泛素分子连接到通道作为一个重要的翻译后调节信号。
The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity inXenopusoocytes. ROMK1 channel activity increased from 8.1 to 27.2 μA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.