Department of Medicine, University of California, Irvine, USA.
Am J Physiol Gastrointest Liver Physiol. 2011 Nov;301(5):G808-13. doi: 10.1152/ajpgi.00269.2011. Epub 2011 Aug 11.
The human thiamine transporter-1 (hTHTR-1) contributes to intestinal thiamine uptake, and its function is regulated at both the transcriptional and posttranscriptional levels. Nothing, however, is known about the protein(s) that may interact with hTHTR-1 and affects its cell biology and physiology. We addressed this issue in the present investigation using a bacterial two-hybrid system to screen a human intestinal cDNA library with the complete coding sequence of hTHTR-1 as a bait. Our results showed that a member of the tetraspanin family of proteins, Tspan-1, interacts with hTHTR-1. Coimmunoprecipitation and glutathione S-transferase (GST)-pulldown assays confirmed the existence of such an interaction between hTspan-1 and hTHTR-1 in human intestinal epithelial Caco-2 cells. Furthermore, live cell confocal imaging demonstrated that hTspan-1 and hTHTR-1 colocalize in human intestinal epithelial HuTu-80 cells. The importance of the interaction between hTspan-1 and hTHTR-1 for cell biology of the thiamine transporter was examined in HuTu-80 cells stably expressing hTHTR-1. Coexpression of hTspan-1 in these cells led to a significant decrease in the rate of degradation of hTHTR-1 compared with cells expressing the hTHTR-1 alone; in fact the half-life of the hTHTR-1 protein was twice longer in the former cell type compared with the latter cell type (12 h vs. 6 h, respectively). This finding was also confirmed at the functional level when a significantly higher thiamine uptake was observed in cycloheximide-treated (6 h) cells expressing hTHTR-1 together with hTspan-1 compared with those expressing hTHTR-1 alone. These studies demonstrate for the first time that Tspan-1 is an interacting partner with hTHTR-1 and that this interaction affects hTHTR-1 stability.
人硫胺素转运蛋白-1（hTHTR-1）有助于肠道硫胺素摄取，其功能受转录和转录后水平的调节。然而，尚不清楚可能与 hTHTR-1 相互作用并影响其细胞生物学和生理学的蛋白质。在本研究中，我们使用细菌双杂交系统，以 hTHTR-1 的完整编码序列作为诱饵，筛选人肠 cDNA 文库，解决了这个问题。我们的结果表明，四跨膜蛋白家族的一个成员 Tspan-1 与 hTHTR-1 相互作用。免疫共沉淀和谷胱甘肽 S-转移酶（GST）下拉实验证实了 hTspan-1 和 hTHTR-1 之间存在这种相互作用在人肠上皮细胞 Caco-2 中。此外，活细胞共聚焦成像显示 hTspan-1 和 hTHTR-1 在人肠上皮细胞 HuTu-80 中共定位。在稳定表达 hTHTR-1 的 HuTu-80 细胞中，研究了 hTspan-1 和 hTHTR-1 之间的相互作用对硫胺素转运体生物学的重要性。在这些细胞中共同表达 hTspan-1 导致 hTHTR-1 的降解率与单独表达 hTHTR-1 的细胞相比显著降低；事实上，hTHTR-1 蛋白的半衰期在前者细胞类型中比后者细胞类型长两倍（分别为 12 h 和 6 h）。当在表达 hTHTR-1 与 hTspan-1 的细胞中观察到在环己酰亚胺处理（6 h）后，硫胺素摄取显著增加时，在功能水平上也证实了这一发现，与单独表达 hTHTR-1 的细胞相比。这些研究首次表明 Tspan-1 是 hTHTR-1 的相互作用伙伴，这种相互作用影响 hTHTR-1 的稳定性。
