Posttranslational modifications of the photoreceptor-specific ABC transporter ABCA4.
Posttranslational modifications of the photoreceptor-specific ABC transporter ABCA4.
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DOI：
10.1021/bi200774w
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发表时间：
2011-08-16
期刊：
影响因子：
2.9
通讯作者：
Palczewski, Krzysztof
中科院分区：
文献类型：
--
作者：
Tsybovsky, Yaroslav;Wang, Benlian;Quazi, Faraz;Molday, Robert S.;Palczewski, Krzysztof
ABCA 4是一种光感受器特异性ATP结合盒转运蛋白，参与光感知过程中视网膜中产生的全反式视黄醇的清除。该蛋白质的多个突变与Stargardt病和其他视觉障碍有关。在这里，我们报告的第一个系统的研究，翻译后修饰的原生ABCA 4纯化牛杆外节。通过质谱分析，在胞质外域1和2中检测到7个N-糖基化位点，证实了先前提出的ABCA 4的拓扑模型。修饰寡糖相对较短且均质，主要代表N-糖基化的高甘露糖类型。在胞质结构域1中检测到5个磷酸化位点，其中4个位于ABCA亚家族成员中保守的接头“调节样”区域。与已发表的结果相反，发现ABCA 4的磷酸化不依赖于光。使用在哺乳动物细胞中异源表达的人ABCA 4突变体，我们发现Stargardt病相关的丙氨酸突变在位置901的磷酸化位点导致蛋白质错误折叠和降解。此外，替换S1317磷酸化位点降低了ABCA 4的基础ATP酶活性，而S1185或T1313磷酸化位点中的丙氨酸突变导致全反式视黄醇刺激的ATP酶活性显著降低，而不影响基础活性、蛋白表达或定位。与此观察结果一致，天然牛ABCA 4的部分去磷酸化导致基础和刺激ATP酶活性的降低。因此，我们提出的第一个证据表明，磷酸化ABCA 4可以调节其功能。
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.
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影响因子：
4.8
作者：
Maeda, Akiko;Maeda, Tadao;Palczewski, Krzysztof
通讯作者：
Palczewski, Krzysztof
影响因子：
4.4
作者：
Maeda, Akiko;Golczak, Marcin;Palczewski, Krzysztof
通讯作者：
Palczewski, Krzysztof
DOI：
10.1016/j.jasms.2007.08.010
发表时间：
2007-11-01
影响因子：
3.2
作者：
Gao, Yuan;Wang, Yinsheng
通讯作者：
Wang, Yinsheng
影响因子：
3.5
作者：
Cideciyan, Artur V.;Swider, Malgorzata;Palczewski, Krzysztof
通讯作者：
Palczewski, Krzysztof
影响因子：
4.8
作者：
Ahn, J;Wong, JT;Molday, RS
通讯作者：
Molday, RS