A Split-Ubiquitin Two-Hybrid Screen for Proteins Physically Interacting with the Yeast Amino Acid Transceptor Gap1 and Ammonium Transceptor Mep2

Several nutrient permeases have been identified in yeast, which combine a transport and receptor function, and are called transceptors. The Gap1 general amino acid permease and the Mep2 ammonium permease mediate rapid activation by amino acids and by ammonium, respectively, of the protein kinase A (PKA) pathway in nitrogen-starved cells. Their mode of action is not well understood. Both proteins are subject to complex controls governing their intracellular trafficking. Using a split-ubiquitin yeast two-hybrid screen with Gap1 or Mep2 as bait, we identified proteins putatively interacting with Gap1 and/or Mep2. They are involved in glycosylation, the secretory pathway, sphingolipid biosynthesis, cell wall biosynthesis and other processes. For several candidate interactors, determination of transport and signaling capacity, as well as localization of Gap1 or Mep2 in the corresponding deletion strains, confirmed a functional interaction with Gap1 and/or Mep2. Also common interacting proteins were identified. Transport and signaling were differentially affected in specific deletion strains, clearly separating the two functions of the transceptors and confirming that signaling does not require transport. We identified two new proteins, Bsc6 and Yir014w, that affect trafficking or downregulation of Gap1. Deletion of EGD2, YNL024c or SPC2 inactivates Gap1 transport and signaling, while its plasma membrane level appears normal.. Vma4 is required for Mep2 expression, while Gup1 appears to be required for proper distribution of Mep2 over the plasma membrane. Some of the interactions were confirmed by GST pull-down assay, using the C-terminal tail of Gap1 or Mep2 expressed in E.coli. Our results reveal the effectiveness of split-ubiquitin two-hybrid screening for identification of proteins functionally interacting with membrane proteins. They provide several candidate proteins involved in the transport and signaling function or in the complex trafficking control of the Gap1 and Mep2 transceptors.

已在酵母中鉴定出多种兼具转运与受体功能的营养物质通透酶，此类蛋白被命名为转运受体（transceptor）。Gap1通用氨基酸通透酶与Mep2铵离子通透酶，可分别介导氮饥饿状态下细胞内蛋白激酶A（PKA）通路被氨基酸与铵离子快速激活，二者的作用机制目前尚未完全阐明。这两种蛋白均受复杂的细胞内运输调控机制所支配。本研究以Gap1或Mep2为诱饵，通过泛素分裂（split-ubiquitin）酵母双杂交筛选技术，鉴定出一批可与Gap1和/或Mep2发生潜在相互作用的蛋白，这些蛋白涉及糖基化、分泌通路、鞘脂生物合成、细胞壁生物合成等多种生物学过程。针对部分候选互作蛋白，通过检测其转运与信号传导能力，以及Gap1或Mep2在对应基因缺失菌株中的定位情况，证实了它们与Gap1和/或Mep2存在功能性相互作用。此外，本研究还鉴定出二者共有的互作蛋白。在特定基因缺失菌株中，转运与信号传导功能受到差异性影响，这一结果清晰区分了转运受体的两项功能，并证实信号传导过程并不依赖于转运活性。本研究还鉴定出两种可调控Gap1运输或其表达下调的新型蛋白：Bsc6与Yir014w。EGD2、YNL024c或SPC2基因缺失会使Gap1的转运与信号传导功能失活，但Gap1在质膜上的表达水平未出现明显异常。Vma4基因对于Mep2的表达不可或缺，而Gup1则似乎是维持Mep2在质膜上正常分布所必需的。部分互作关系通过GST pull-down实验得到了验证：实验采用在大肠杆菌（E. coli）中表达的Gap1或Mep2 C末端尾段作为检测底物。本研究结果证实，泛素分裂双杂交筛选技术可有效用于鉴定与膜蛋白发生功能性相互作用的蛋白。本研究为解析Gap1与Mep2这两种转运受体的转运、信号传导功能及其复杂的运输调控机制，提供了一批候选蛋白。