Article Figures & Data
Figures
- Fig 1
BUL genes are not essential for viability but are important for response to heat stress. (A) HA epitope-tagged Bul3p was expressed in a bul3Δ null mutant strain, and the protein was visualized by immunoblotting using anti-HA antibodies. The arrowheads indicate two major translation products of 55 to 60 kDa; the asterisk indicates a readthrough translation product of ∼100 kDa. (B) HABul3p was expressed in a bul3Δ strain, along with a Y100A (HABul3ΔPY) form, a variant that lacks the internal stop codon (HABul3ns), and an empty HA vector control. Extracts from log-phase cells (Input) were subjected to immunoprecipitation with anti-HA affinity resin (IP αHA) and subjected to immunoblotting with anti-HA and anti-Rsp5 antibodies. (C) Tenfold serial dilution of yeast strains with the BUL genes deleted alone (Δ1, Δ2, and Δ3), in pairs (Δ1,2; Δ1,3; and Δ2,3), or in combination (Δ1,2,3) were grown for 3 days at either 30°C or 42°C. WT, wild type. (D) Serial deletions as described for panel C with strains expressing HABul3 or HABul3ΔPY constructs. (E and F) In vitro ubiquitination assays using recombinant S-tagged Bul1p and Bul2p and large (Bul3ns) and small (Bul3stop) forms of Bul3p. The reactions were performed with methylated ubiquitin (Ub) using equal quantities, as determined using immunoblots with anti-Rsp5p antibodies (F), of wild-type Rsp5p and variants that had only a single functional WW domain remaining (WW3, WW2, and WW1) before being subjected to immunoblotting with anti-S-tag antibodies.
- Fig 2
BUL gene deletions show complex growth phenotypes but are not required for ubiquitin-mediated processing of Mga2p. (A) Tenfold serial dilution of BUL gene deletion strains grown on YPD agar medium, YPD plus 25 μM CdCl2 (Cd) or 3 μM phleomycin (Phl), or YEP plus 2% (vol/vol) ethanol (EtOH). (B) Tenfold serial dilution of BUL gene deletion strains grown on YPD agar medium plus 1 mM AZC, synthetic complete medium with no arginine (SC), or SC minus Arg plus 14 μM canavanine (Can). (C) Immunoblot using anti-HA antibodies of HA-Mga2p processing from P120 to P90 in wild-type and BUL gene deletion (Δ1,2,3) strains.
- Fig 3
Sorting of mCherryΔNSmf1p in metal-depleted medium following the addition of CdCl2. (A) mCherry fluorescence and differential interference contrast microscopy images of wild-type and single BUL gene deletion strains expressing mCherryΔNSmf1p following growth on metal-depleted media (−metal) and 2 h after the addition of 25 μM CdCl2. The arrowheads indicate plasma membrane localization. (B) (Top) Immunoblots against Tap-tagged ΔNSmf1p in total protein extracts from wild-type and bul1Δ (Δ1) and bul2Δ (Δ2) strains. (Bottom) PonceauS staining was used as a loading control.
- Fig 4
Sorting of GFPGap1p in nitrogen-poor medium. Shown are GFP fluorescence and differential interference contrast microscopy images of wild-type and single BUL gene deletion strains expressing GFPGap1p following growth in medium with proline as the sole nitrogen source.
- Fig 5
Sorting of GFPCan1p following growth in excess arginine. Shown are GFP fluorescence and differential interference contrast microscopy images of wild-type and single BUL gene deletion strains expressing GFPCan1p. The images were obtained before (−Arg) and 2 h after (+Arg) the addition of 100 mg/liter arginine to log-phase cells grown in arginine-free synthetic complete medium. The arrowheads indicate plasma membrane localization.
