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首页> 外文期刊>Biochemistry >Posttranslational Modifications of the Photoreceptor-Specific ABC Transporter ABCA4
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Posttranslational Modifications of the Photoreceptor-Specific ABC Transporter ABCA4

机译：受体特异性ABC转运蛋白ABCA4的翻译后修饰

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摘要

ABCA4 is a photoreceptor-specific ATP-binding cassette transporter implicated in the clearance of all-trans-retinal produced in the retina during light perception. Multiple mutations in this protein have been linked to Stargardt disease and other visual disorders. Here we report the first systematic study of posttranslational modifications in native ABCA4 purified from bovine rod outer segments. Seven N-glycosylation sites were detected in exocytoplasmic domains 1 and 2 by mass spectrometry, confirming the topological model of ABCA4 proposed previously. The modifying oligosaccharides were relatively short and homogeneous, predominantly representing a high-mannose type of N-glycosylation. Five phosphorylation sites were detected in cytoplasmic domain 1, with four of them located in the linker “regulatory-like” region conserved among ABCA subfamily members. Contrary to published results, phosphorylation of ABCA4 was found to be independent of light. Using human ABCA4 mutants heterologously expressed in mammalian cells, we showed that the Stargardt disease-associated alanine mutation in the phosphorylation site at position 901 led to protein misfolding and degradation. Furthermore, replacing the S1317 phosphorylation site reduced the basal ATPase activity of ABCA4, whereas an alanine mutation in either the S1185 or T1313 phosphorylation site resulted in a significant decrease in the all-trans-retinal-stimulated ATPase activity without affecting the basal activity, protein expression, or localization. In agreement with this observation, partial dephosphorylation of native bovine ABCA4 led to reduction of both basal and stimulated ATPase activity. Thus, we present the first evidence that phosphorylation of ABCA4 can regulate its function.
机译：ABCA4是一种光感受器特异性ATP结合盒转运蛋白，与光感知过程中视网膜中产生的全反式视网膜的清除有关。该蛋白的多个突变与Stargardt病和其他视觉障碍有关。在这里，我们报告从牛杆外段纯化的天然ABCA4中翻译后修饰的第一个系统研究。质谱法在胞质域1和2中检测到7个N-糖基化位点，证实了先前提出的ABCA4拓扑模型。修饰的寡糖相对较短且均匀，主要代表高甘露糖型的N-糖基化。在胞质结构域1中检测到五个磷酸化位点，其中四个位于ABCA亚家族成员之间保守的接头“调控样”区域。与已发表的结果相反，发现ABCA4的磷酸化与光无关。使用在哺乳动物细胞中异源表达的人ABCA4突变体，我们发现在位置901的磷酸化位点与Stargardt病相关的丙氨酸突变导致蛋白质错误折叠和降解。此外，替换S1317磷酸化位点会降低ABCA4的基础ATPase活性，而S1185或T1313磷酸化位点中的丙氨酸突变会导致全反式视网膜刺激的ATPase活性显着降低，而不会影响基础活性蛋白表达或本地化。与该观察结果一致，天然牛ABCA4的部分去磷酸化导致基础和刺激的ATP酶活性降低。因此，我们提供了ABCA4磷酸化可以调节其功能的第一个证据。

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