Heterodimerization of y+LAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy
Heterodimerization of y+LAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy
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DOI：
10.1016/j.bbamem.2007.04.020
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发表时间：
2007-10-01
影响因子：
3.4
通讯作者：
Heiskanen, Kaisa M.
中科院分区：
文献类型：
--
作者：
Kleemola, Maaria;Toivonen, Minna;Heiskanen, Kaisa M.
y(+)LAT-1和4F2hc是阳离子氨基酸转运蛋白复合物的亚基，主要位于小肠和肾小管上皮细胞的基底外侧质膜中。 y+LAT-1 突变会损害该复合物的转运功能，并导致选择性氨基酸尿、赖氨酸尿蛋白不耐受（LPI，OMIM #222700），与严重、复杂的临床症状相关。活性转运蛋白的亚基共定位于质膜中，但二聚化的确切过程尚不清楚，因为尚未获得该转运蛋白在完整人体细胞中组装的直接证据。在本研究中，我们使用荧光共振能量转移（FRET）显微镜来研究表达与 ECFP 或 EYFP 融合的 y(+)LAT 1 和 4F2hc 的 HEK293 细胞中 y(+)LAT 1 和 4F2hc 的相互作用。通过测量受体 (EYFP) 光漂白后供体荧光团 (ECFP) 的荧光强度变化来量化 FRET。从内质网和高尔基复合体到质膜，整个细胞都可以检测到增加的供体荧光。因此，我们的数据证明了 y(+)LAT 1 和 4F2hc 在质膜之前的相互作用，从而为 4F2hc 作为伴侣协助 y(+)LAT-1 转运至质膜提供了证据。 (C) 2007 Elsevier B.V. 保留所有权利。
y(+)LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y+LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y(+)LAT 1 and 4F2hc in HEK293 cells expressing y(+)LAT 1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y(+)LAT 1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y(+)LAT-1 to the plasma membrane. (C) 2007 Elsevier B.V. All rights reserved.