FIG 8.

Arrestin-like adaptors of Rsp5 promote substrate-induced downregulation of Gap1 and Can1. (A) (Top) gap1Δ (EK008) and gap1Δ bul1Δ bul2Δ (JA493) cells expressing Gap1-112–GFP (pMA074) were grown on Gal Pro medium before addition of glucose for 0.5 h. Phe (5 mM) was then added for 2 h. (Bottom) The same cells and growth conditions, except that Phe was added for 0.5 h. Crude cell extracts were then prepared and immunoblotted with anti-GFP antibodies. (B) Wild-type (23344c) and gap1Δ art1Δ (JA937) cells expressing GFP-fused Can1 (pKG036) or Can1(S176N-T456S) (pKG066) were grown on glucose Am medium (hence, Gap1 synthesis is repressed in the wild type). Arg or Lys was then added for 3 h before analysis by fluorescence microscopy. (C) (Top) gap1Δ can1 (ES006) and gap1Δ can1 rsp5(npi1) (35237c) cells expressing Art1-HA under the natural promoter of the ART1 gene were grown on glucose Pro medium. Crude cell extracts were then prepared and immunoblotted with anti-HA antibodies. (Bottom) gap1Δ can1 ART1-HA cells (ES006) transformed with a CAN1 plasmid (pKG036) were grown on glucose Pro medium. Arg (5 mM) or Am (20 mM) was added for 20 min, and crude cell extracts were prepared and immunoblotted with anti-HA antibodies. (D) (Top) gap1Δ cells (EK008) expressing Gap1-112–GFP (pMA074) were grown on Gal Pro medium before addition of glucose for 0.5 h (left), Am (20 mM) for 2 h (middle), and Phe (5 mM) for 2 h (right) to half of the culture. (Bottom) Cells expressing Bul2-HA under the natural promoter of the BUL2 gene (MA032) and Gap1-112–GFP (pMA074) were grown on Gal Pro medium and treated as described above for the indicated time intervals. Crude cell extracts were prepared after the indicated times and immunoblotted with anti-HA antibodies.