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. 2020 Mar 2;9:e51065. doi: 10.7554/eLife.51065

Figure 6. The Cdc48 co-factors Ubx2 and Doa1 are implicated in MAD.

(A) CHX chase for the indicated times at 37°C assessing the turnover of sam35-2HAts (pMM157) and sen2-1HAts (pMM160) in WT (BY4741) and doa1Δ cells (yJS208). Proteins were detected by immunoblotting. Graphed below is the mean and SD of the PGK-normalized HA signal at each time point for three biological replicates. (B) CHX chase as in A for the indicated times at 37°C assessing the turnover of sam35-2HAts (pMM157) and sen2-1HAts (pMM160) in WT (BY4741) and ubx2Δ cells (yJS155). (C) CHX chase as in A for the indicated times at 37°C assessing the turnover of sam35-2HAts (pMM157) and sen2-1HAts (pMM160) in WT (BY4741) and ubx2Δ (yJS155) cells co-expressing either empty vector (EV; pRS315) or CEN Ubx2-FLAG (pMM242). (D) CHX chase as in A for the indicated times at 37°C assessing the turnover of sam35-2HAts (pMM231) or sen2-1HAts (pMM234) in WT (BY4741) cells or ubx2Δ (yJS155) cells expressing either EV (pRS315) or Doa1-FLAG (pMM254) from a high copy 2μ plasmid. (E) Ubiquitination of sam35-2HAts and sen2-1HAts was assessed by IP using TUBE agarose or anti-HA agarose from ubx2Δ (yJS155) and WT (BY4741) lysates expressing EV (pRS315), sam35-2HAts (pMM157), or sen2-1HAts (pMM160), followed by immunoblotting with HA or c-myc antibody. 2.5% or 1% of the IP input lysate was reserved and analyzed by immunoblotting. (F) Co-IP of Ubx2-FLAG (pMM242) with sam35-2HAts or sen2-1HAts (pMM231 and 234, respectively) from pre1-1 pre2-2 (WCG4-11/21a) cells was assessed by immunoblotting with the indicated antibodies. IP of Ubx2-FLAG from cells co-expressing EV (pRS316) in place of HA-tagged substrates and 0.5% of the input lysate are shown for comparison. (G) Lysate (‘Total’) and increasing amounts of mitochondria purified by 12,000xg and sucrose gradient fractionation (‘Mitos’) from Ubx2-TAP-expressing cells were examined by immunoblotting with the indicated antibodies. (H) Lysates from WT (BY4741) and ubx2Δ (yJS155) cells expressing sam35-2HAts or sen2-1HAts (pMM157 and 160, respectively) were fractionated at 12,000xg at 37°C into mitochondrial pellets (P) and post-mitochondrial supernatants (S). Fractions were subject to immunoblotting with antibodies to HA, PGK, and PORIN.

Figure 6—source data 1. Quantifications of cycloheximide chases.

Figure 6.

Figure 6—figure supplement 1. The Cdc48 co-factors Ubx2 and Doa1 are required for MAD.

Figure 6—figure supplement 1.

(A) CHX chase for the indicated times at 37°C assessing the turnover of sam35-2HAts or sen2-1HAts (pMM157 or 160, respectively) in vms1Δ compared to isogenic WT (BY4741). Proteins were detected by immunoblotting. Percentage of each ts- substrate remaining is labeled for each time point under blots. (B) CHX chase for the indicated times at 37°C assessing the turnover of sam35-2HAts or sen2-1HAts (pMM157 or 160, respectively) in the indicated UBX protein deletion strains compared to isogenic WT (BY4741). (C) CHX chase for the indicated times at 37°C assessing the turnover of SAM35HA or SEN2HA (pMM158 or 159, respectively) in ubx2Δ (yJS155) compared to isogenic WT (BY4741). (D) Co-IP of Ubx2-FLAG (pMM242) with SAM35HA, sam35-2HAts, SEN2HA, or sen2-1HAts (pMM232, pMM231, pMM233, and 234, respectively) from pre1-1 pre2-2 (WCG4-11/21a) cells was assessed by immunoblotting with the indicated antibodies. IP of Ubx2-FLAG from cells co-expressing EV (pRS316) in place of HA-tagged substrates and 0.5% of the input lysate are shown for comparison. (E) CHX chase for the indicated times at 30°C assessing the turnover of Fzo1HA (pADH1-Fzo1pHA) in WT (BY4741) and doa1Δ (yJS208) strains. (F) CHX chase for the indicated times at 30°C assessing the turnover of Fzo1HA (pADH1-Fzo1pHA) in WT (BY4741) and ubx2Δ (yJS155) cells co-expressing either EV (pRS315) or Ubx2-FLAG (pMM242). (G) Co-IP of Ubx2-FLAG (pMM242) with Fzo1HA (pADH1-Fzo1pHA) from pre1-1 pre2-2 (WCG4-11/21a) cells was assessed by immunoblotting with the indicated antibodies. Negative control of IP of Ubx2-FLAG from cells co-expressing empty vector (pRS316) in place of HA-tagged substrates is shown, as is 0.5% of the input lysate. (H) Ubiquitination of Fzo1HA was assessed by IP using TUBE agarose from ubx2Δ (yJS155) and WT (BY4741) lysates expressing EV (pRS316) or Fzo1HA (pADH1-Fzo1pHA) followed by immunoblotting with HA antibody. 2.5% of the TUBE input lysate was reserved and analyzed by immunoblotting. (I) Microscopy analysis of immobilized Ubx2-GFP cells co-expressing either mtRFP (pMD12) to label mitochondria or SEC63-RFP (pSM1959) to label the ER. Some regions of co-localization are highlighted with yellow arrows. ‘Merge’ of GFP (green) and RFP (magenta) channels and differential interference contrast (DIC) are shown; scale bar = 10 um. (J) CHX chase for the indicated times at 37°C assessing the turnover of sam35-2HAts or sen2-1HAts (pMM157 or 160, respectively) in WT (BY4741) and mmm1Δ, mdm12Δ, mdm34Δ, and mdm10Δ strains. (K, L) CHX chase for the indicated times at 37°C assessing the turnover of sam35-2HAts or sen2-1HAts (pMM157 or 160, respectively) in a ubc6Δubc7Δ (K; SM5364) or doa10Δhrd1Δ (L, SM5360) strains relative to their isogenic WT strain (BY4741). (M) CHX chase for the indicated times at 30°C assessing the turnover of Fzo1HA (pADH1-Fzo1pHA) in WT (BY4741) and mmm1Δ, mdm12Δ, mdm34Δ, and mdm10Δ strains. (N) Lysates from WT (BY4741) and ubx2Δ (yJS155) cells expressing Fzo1HA (pMM190) were fractionated at 12,000xg at 37°C into mitochondrial pellets (P) and post-mitochondrial supernatants (S). Fractions were subject to immunoblotting with antibodies to HA, PGK, and PORIN.