A split-ubiquitin two-hybrid screen for proteins physically interacting with the yeast amino acid transceptor Gap1 and ammonium transceptor Mep2.

Abstract

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.