(A) Silver-stained SDS polyacrylamide gel showing affinity purified Ubp2-TAP and Rup1-TAP protein complexes. Arrows indicate polypeptides identified by gel band excision followed by MALDI-ToF mass spectrometry. The asterisk indicates degradation products of Ubp2 and Rup1. (B) Ubp2 recovery from Ubp2-TAP, Ubp2-TAP rup1Δ, and Rup1-TAP strains. Arrow indicates gel band identified as Ubp2 by MALDI-ToF mass spectrometry. (C) Co-immunoprecipitation of a fraction of cellular Rsp5 with Ubp2 and Rup1. Cell extracts were depleted of endogenously TAP-tagged Ubp2 or Rup1 with IgG (IP lanes), and the remaining soluble Rsp5 (post-IP lanes) and bait proteins probed by Western blot with anti-Rsp5 antibodies which also recognized the protein A portion of the TAP tag. The ‘input’ lane shows the Rsp5 level in the extract prior to IP. (D) Rup1 tethers Ubp2 to Rsp5. IgG-based immunoprecipitation of Rup1-TAP and Ubp2-TAP from WT, ubp2Δ or rup1Δ strains. The co-purification of Rsp5 with the baits was determined by Western blotting using anti-Rsp5 antibodies. (E) Plasmids expressing FLAG(F/H)-tagged wildtype Rup1 or the Y135F point mutant were transformed into cells lacking endogenous Rup1. After immunoprecipitation with anti-FLAG antibodies, Rup1 and Rsp5 were detected by Western blotting. The bottom panel show the expression levels of Rsp5 in whole cell extracts by Western blotting against Rsp5.

Wildtype, ubp2Δ, vps37Δ, fur4Δ single mutants, and vps37Δubp2Δ double mutant cells were transformed with a URA3 plasmid and subsequently tested for sensitivity to 5-FU. Exponentially growing cells were serially diluted 10-fold (starting OD₆₀₀ ∼0.7) and spotted onto SD plates with 5 µM 5-FU. Plates were imaged either after 4 days (left panel) or 7 days (right panel).

pFUR4-GFP and a plasmid overexpressing ubiquitin (+) or an empty plasmid (−) were transformed into cells. Strains were grown in sucrose, and diluted to OD₆₀₀ = 0.5 in media containing galactose and 0.1 mM copper sulphate (to overexpress ubiquitin) and grown for 4 hours. Fur4 transcription was stopped by adding glucose for 1 hour to chase Fur4 to the plasma membrane. Uracil (40 µg/ml) was then added to induce Fur4 internalization. The GFP signal was viewed by fluorescence confocal microscopy before (−) and at 60 min after (+) uracil addition. Note that the left panel contains the same images as Figure 7, as the experiments were done in parallel.

pFUR4-GFP or pUb-FUR4-GFP expression plasmids were transformed into WT and ubp2Δ cells. Strains were grown in sucrose, diluted (OD₆₀₀ = 0.5) in media containing galactose and Fur4 synthesis induced for 4 hours. Transcription was stopped by adding glucose for 1 hour to chase Fur4 fusion reporters to the plasma membrane. Uracil (40 µg/ml) was then added to trigger internalization. GFP signal was viewed by fluorescence confocal microscopy both before (T0) and 60 min after uracil addition. (Note: cells in the left panel were also transformed with a control vector (YEp46Δ) and incubated with 0.1 mM copper sulphate, as the experiment was done in parallel to that shown in Figure 5.)

(A, B, D) The indicated over-expression plasmids, driven by a galactose inducible promoter, were transformed into either wildtype or mutant strains. Cell cultures were then serially diluted 10-fold (starting OD₆₀₀ of 0.5) and spotted onto SC-URA media containing glucose (non-inducing) or galactose (inducing). (B) Growth of rsp5-326 mutant cells bearing pHA-RSP5 (a low copy, but not galactose responsive plasmid), and/or a second overexpression plasmid as indicated. (C) Growth of cells deleted for either UBP2 and RUP1 alone, or in combination with an RSP5 temperature sensitive allele (rsp5-1), on rich media (YPD) at either 30°c (permissive for rsp5-1) or 34°c (semi-permissive for rsp5-1).

A FUR4-GFP fusion reporter was transformed into cells, which were then grown in raffinose overnight. Galactose was added (OD₆₀₀ = 0.6) for 2 hours to induce synthesis, followed by glucose for 10 min to chase Fur4-GFP to the plasma membrane, and then uracil (40 µg/ml) to trigger transporter internalization. (A) Fur4-GFP signal viewed by fluorescence microscopy at the indicated time points (min) after uracil addition. T0 indicates cells immediately before addition. (B) Fur4-GFP protein levels as measured by Western blotting. Total protein extracts were generated from cells harvested at the indicated time points following uracil addition. Fur4-GFP was probed with anti-GFP antibodies. 3-phosphoglycerate kinase (PGK) was used as a loading control. The asterisk indicates an unknown background band.

(A) pFUR4-GFP bearing cells were grown in raffinose overnight. Galactose was added for 2 hours to induce synthesis, and glucose was then added for 10 min to chase Fur4-GFP to the plasma membrane. CHX (0.1mg/ml) was added and GFP signal examined by fluorescence microscopy and Nomarski optics. Time refers to the time after addition of CHX, with 0 min as pre-induction. (B) Uracil uptake was measured at different times after the addition of CHX in WT and ubp2Δ strains. Results are expressed as a percentage of the initial uracil uptake, and plotted on a log scale. (C) Fur4 ubiquitin profile at the plasma membrane is unchanged in ubp2Δ mutants. pFUR4 was transformed into WT, ubp2Δ, rup1Δ, and rsp5-1, and cells, which were grown in raffinose overnight. Expression from pFUR4 was induced for 90 min with galactose before adding glucose for 15min to chase Fur4 to the plasma membrane. Total protein extracts (lysate) and enriched membrane fractions were collected and analyzed by Western blotting to visualize Fur4 and Fur4-ubiquitin conjugates. 3-phosphoglycerate kinase (PGK) and porin, a mitochondrial membrane protein, were used as loading controls.

α-factor (5 ul of 100 µM, 40 µM or water alone) was spotted onto sterile filter disks, and placed onto solid media containing WT, ubp2Δ, rup1Δ, or end3Δ MATa cells. Plates were then incubated at 30°C for one day and imaged. Halo diameters (mm) were measured and averaged from five replicates of 100 µM alpha factor spots.