Department of Immunobiology, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Shiwa-gun, Iwate 028-3694, Japan.
Biochem J. 2013 Jun 15;452(3):467-75. doi: 10.1042/BJ20130115.
Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4-HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.
秀丽隐杆线虫 HAF-4 和 HAF-9 是高度同源的人类溶酶体肽转运蛋白 TAPL [TAP（抗原加工相关转运体）样；ABCB9]的半 ABC（ATP 结合盒）转运体。我们之前报道过，HAF-4 和 HAF-9 都定位于肠道细胞器的一部分膜上，并且是这些细胞器形成和其他生理方面所必需的。在本文中，我们报告了 HAF-4 和 HAF-9 之间的遗传和物理相互作用。HAF-4 和 HAF-9 的过表达分别不能挽救 haf-9 和 haf-4 缺失突变体的肠道细胞器缺陷，表明它们不能相互替代。双 haf-4 和 haf-9 突变体的表型并不比单突变体更严重，表明它们具有协同功能。免疫沉淀实验证明了它们的物理相互作用。本研究的结果表明，HAF-4 和 HAF-9 形成异二聚体。此外，对 GFP（绿色荧光蛋白）标记的 HAF-4 或 HAF-9 转基因线虫的缺失突变体和 RNAi（RNA 干扰）敲低实验的 Western blot 分析表明，HAF-4-HAF-9 异二聚体的形成是其稳定所必需的。这些发现为 ABC 转运体如何采用稳定的功能形式提供了线索。
