Kleemola Maaria, Toivonen Minna, Mykkänen Juha, Simell Olli, Huoponen Kirsi, Heiskanen Kaisa M
Department of Medical Genetics, University of Turku, Turku, Finland; Turku Centre for Biotechnology, University of Turku, Biocity, Turku, Finland.
Biochim Biophys Acta. 2007 Oct;1768(10):2345-54. doi: 10.1016/j.bbamem.2007.04.020. Epub 2007 May 3.
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.
y(+)LAT-1和4F2hc是一种阳离子氨基酸转运复合物的亚基，主要位于小肠和肾小管上皮细胞的基底外侧质膜。y(+)LAT-1的突变会损害该复合物的转运功能，并导致选择性氨基酸尿症、赖氨酸尿性蛋白不耐受症（LPI，OMIM #222700），伴有严重、复杂的临床症状。活性转运体的亚基在质膜中共定位，但由于尚未获得该转运体在完整人类细胞中组装的直接证据，二聚化的确切过程尚不清楚。在本研究中，我们使用荧光共振能量转移（FRET）显微镜来研究在表达与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）的荧光强度变化来定量FRET。从内质网、高尔基体复合物到质膜，整个细胞中都能检测到供体荧光增加。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作为伴侣蛋白协助y(+)LAT-1转运到质膜提供了证据。
