Fig. 2.

Subcellular localization of Gtr1 and Gtr2. (A) gtr1-gfp and gtr2-rfp cells were grown in EMM at 30°C in the presence and in the absence of amino acids. Gtr1–GFP and Gtr2–RFP localized to structures similar to the vacuolar membranes, independently of the presence of amino acids. (B) gtr1-gfp cells were grown in EMM at 30°C, and FM4-64 staining was used to identify vacuoles and corroborate that Gtr1–GFP localized to the vacuole membranes. (C) Gtr1–GFP and Gtr2–RFP colocalized to the vacuolar surface in the presence and in the absence of amino acids. (D) gtr1-flag, gtr2-rfp and gtr1-flag gtr2-rfp cells were grown in EMM at 30°C and transferred into EMM in the presence and in the absence of amino acids. Exponentially growing cells were collected after 1 hour and cell lysates were pulled down using anti-Flag M2 beads. Gtr1–Flag physically interacted with Gtr2–RFP and this interaction increased in the presence of amino acids. The relative interaction between Gtr1–Flag and Gtr2–RFP was estimated by densitometry using ImageJ software and normalized to the amount of Gtr2–RFP pulled down with anti-Flag antibodies in the presence of amino acids, which was set as 1. Scale bars: 10 μm.