Identification of Cysteine Residues in Human Cationic Amino Acid Transporter hCAT-2A That Are Targets for Inhibition by N-Ethylmaleimide
Identification of Cysteine Residues in Human Cationic Amino Acid Transporter hCAT-2A That Are Targets for Inhibition by N-Ethylmaleimide
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DOI：
10.1074/jbc.m113.490698
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发表时间：
2013-10-18
影响因子：
4.8
通讯作者：
Closs, Ellen I.
中科院分区：
文献类型：
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作者：
Beyer, Sarah R.;Mallmann, Robert T.;Closs, Ellen I.
在大多数细胞中，阳离子氨基酸（例如 L-精氨酸、L-赖氨酸和 L-鸟氨酸）由阳离子 (CAT) 和 y(+)L (y(+)LAT) 氨基酸转运蛋白转运。在人红细胞中，半胱氨酸修饰剂 N-乙基马来酰亚胺 (NEM) 已被证明可以抑制系统 y(+)（最有可能是 CAT-1），但不能抑制系统 y(+)L（Deves, R.、Angelo, S. 和 Chavez, P. (1993) J. Physiol. 468, 753-766）。因此，我们想知道对 NEM 的敏感性是否可以区分所有 CAT 和 y(+)LAT 同工型。在非洲爪蟾卵母细胞中进行的转运测定表明，实际上所有人类 CAT（包括低亲和力 hCAT-2A），但 y(+)LAT 亚型均不受 NEM 抑制。 hCAT-2A 抑制并非由于质膜中转运蛋白表达减少所致，表明 NEM 降低了内在转运蛋白活性。所有 CAT 同工型中保守的七个半胱氨酸残基中每一个的单独突变不会导致 hCAT-2A 的 NEM 不敏感性。然而，一种缺乏半胱氨酸的突变体不再被 NEM 抑制，这表明抑制是通过 hCAT-2A 中多个半胱氨酸的修饰而发生的。事实上，双突变体 C33A/C273A 对 NEM 抑制也不敏感，而在不含半胱氨酸的突变体中在 33 或 273 位重新引入半胱氨酸会导致 NEM 敏感性。因此，我们确定 hCAT-2A 中的 Cys-33 和 Cys-273 作为 NEM 抑制的靶标。此外，所有具有 Cys-33 突变的蛋白质均表现出转运活性显着降低，这表明，令人惊讶的是，位于细胞质 N 末端的该残基对于转运蛋白功能非常重要。
In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y(+)L (y(+)LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y(+) (most likely CAT-1), but not system y(+)L (Deves, R., Angelo, S., and Chavez, P. (1993) J. Physiol. 468, 753-766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y(+)LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y(+)LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced transporter expression in the plasma membrane, indicating that NEM reduces the intrinsic transporter activity. Individual mutation of each of the seven cysteine residues conserved in all CAT isoforms did not lead to NEM insensitivity of hCAT-2A. However, a cysteine-less mutant was no longer inhibited by NEM, suggesting that inhibition occurs through modification of more than one cysteine in hCAT-2A. Indeed, also the double mutant C33A/C273A was insensitive to NEM inhibition, whereas reintroduction of a cysteine at either position 33 or 273 in the cysteine-less mutant led to NEM sensitivity. We thus identified Cys-33 and Cys-273 in hCAT-2A as the targets of NEM inhibition. In addition, all proteins with Cys-33 mutations showed a pronounced reduction in transport activity, suggesting that, surprisingly, this residue, located in the cytoplasmic N terminus, is important for transporter function.