Glycosylation Regulates Prestin Cellular Activity
Glycosylation Regulates Prestin Cellular Activity
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DOI：
10.1007/s10162-009-0196-5
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发表时间：
2010-03-01
影响因子：
2.4
通讯作者：
Pereira, Fred A.
中科院分区：
文献类型：
--
作者：
Rajagopalan, Lavanya;Organ-Darling, Louise E.;Pereira, Fred A.
糖基化是一种常见的蛋白质翻译后修饰，涉及多种细胞功能，包括蛋白质折叠、降解、分选和运输以及膜蛋白再循环。膜蛋白普雷斯廷是外毛细胞（OHC）中基于膜的运动驱动电运动变化（电运动）的重要组成部分，外毛细胞是听觉信号转导的中枢过程。普雷斯廷较早被鉴定为具有两个N-糖基化位点（N163、N166），当突变时，所述N-糖基化位点略微影响培养细胞中的普雷斯廷非线性电容（NLC）功能。在这里，我们表明，双突变体普雷斯廷（NN 163/166 AA）是没有糖基化，并显示出预期的NLC性质在未经处理和胆固醇耗尽HEK 293细胞模型。此外，与容易形成寡聚体的WT普雷斯廷不同，普雷斯廷（NN 163/166 AA）作为单体富集，并且在质膜中更具移动的，表明普雷斯廷的寡聚化依赖于糖基化，但对于HEK 293细胞中NLC的生成不是必需的。然而，在存在增加的膜胆固醇的情况下，与WT普雷斯廷所见的NLC中的超极化移位不同，表达普雷斯廷的细胞（NN 163/166 AA）表现出线性电容功能。为了解释这一发现，我们发现WT普雷斯廷和普雷斯廷（NN 163/166 AA）均参与胆固醇依赖性细胞运输。与WT普雷斯廷相反，普雷斯廷（NN 163/166 AA）显示细胞表面表达的显著胆固醇依赖性降低，这可以解释NLC功能的丧失。基于我们的观察，我们得出结论，糖基化调节普雷斯廷（NN 163/166 AA）的自缔合和细胞运输。这些观察结果是第一次暗示细胞运输和分选在普雷斯廷功能中的调节作用。我们推测，胆固醇的普雷斯廷的调节发生在通过网格蛋白和小窝蛋白依赖的机制，从膜微区的本地化和内化。
Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by clathrin- and caveolin-dependent mechanisms.