1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

Cdc48 regulates a deubiquitylase cascade critical for mitochondrial fusion

  1. Tânia Simões
  2. Ramona Schuster
  3. Fabian den Brave
  4. Mafalda Escobar-Henriques  Is a corresponding author
  1. University of Cologne, Germany
  2. Max Planck Institute of Biochemistry, Am Klopferspitz 18, Germany
Research Article
Cite this article as: eLife 2018;7:e30015 doi: 10.7554/eLife.30015
9 figures, 4 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Cdc48 regulates Fzo1 and mitochondrial fusion.

(A) Mitochondrial morphology of CDC48 mutant cells. Wild-type (wt) or cdc48-2 mutant cells were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid. Cellular (Nomarski) and mitochondrial (GFP) morphology were visualized by fluorescence microscopy. Bottom panel, quantification of four independent experiments (with more than 200 cells each) including mean and standard deviation (SD), as described (Cumming et al., 2007). (B) Ubiquitylation of Fzo1 upon mutation of CDC48. Crude mitochondrial extracts from wt or cdc48-2 mutant cells expressing HA-Fzo1, or the corresponding empty vector, were solubilized and analyzed by SDS-PAGE and immunoblotting using HA-specific antibodies. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated by a black arrowhead or black arrows, respectively. Ubiquitylated forms of Fzo1 are labeled with Ub. Bottom panel, quantification of three independent experiments, normalized to PoS and including SD. **, p≤0.01 (paired t-test). (C) Steady state levels of Fzo1 upon mutation of CDC48. Total cellular extracts of wt or cdc48-2 mutant cells were analyzed by SDS-PAGE and immunoblotting using Fzo1- or Ubc6-specific and, as a loading control, Tom40-specific antibodies. Bottom panels, quantification of three independent experiments, including SD. (D) Proteasome dependence of Fzo1 degradation in cdc48-2 mutant cells. The turnover of endogenous Fzo1 expressed in Δpdr5 Δsnq2 and Δpdr5 Δsnq2 cdc48-2 cells was assessed after inhibition of cytosolic protein synthesis with cycloheximide (CHX), for the indicated time points in exponentially growing cultures in absence or presence of the proteasomal inhibitor MG132. Samples were analyzed by SDS-PAGE and immunoblotting using Fzo1-specific, Ubc6-specific (as an unstable protein control) and Sec61-specific (as a loading control) antibodies. Right panel, quantification of five independent experiments, including SD. PoS, PonceauS staining.

https://doi.org/10.7554/eLife.30015.003
Figure 1—figure supplement 1
Cdc48 regulates Fzo1 and mitochondrial fusion.

(A) Steady state levels of Fzo1 upon mutation of CDC48. Total cellular extracts of Δfzo1 or wt cells or different CDC48 mutant cells were analyzed by SDS-PAGE and immunoblotting using Fzo1-, Ubc6- and Tom40-specific antibodies. Bottom panels, quantification of five independent experiments, including SD. ns, p>0.05; *, p≤0.05; ***, p≤0.001 (One-way ANOVA, Tukey’s multiple comparison test). (Β) Role of Cdc48 cofactors in the steady state levels of Fzo1. Total cellular extracts of wt cells or ufd1-2 and npl4-1 mutant cells were analyzed by SDS-PAGE and immunoblotting using Fzo1- or Ubc6-specific antibodies. Bottom panels, quantification of seven (ufd1-2) or nine (npl4-1) independent experiments, including SD. **p≤0.01; ***p≤0.001 (paired t-test). (C) Steady state levels of Fzo1 upon deletion of DOA1. Total cellular extracts of Δfzo1, wt or Δdoa1 cells were analyzed by SDS-PAGE and immunoblotting using Fzo1-, Ubc6- and Tom40-specific antibodies. Bottom panel, quantification of five independent experiments, including SD. *p≤0.05 (paired t-test). PoS, PonceauS staining.

https://doi.org/10.7554/eLife.30015.004
Figure 1—figure supplement 2
Cdc48 regulates Fzo1 and mitochondrial fusion.

(A) Rescue analysis of Fzo1 steady state levels in cdc48-2 cells. Total cellular extracts of wt or cdc48-2 mutant cells expressing Cdc48, Cdc48A547T or the corresponding empty vector were analyzed by SDS-PAGE and immunoblotting using an HA-specific antibody. (B) Rescue analysis of Fzo1 ubiquitylation in cdc48-2 cells. Crude mitochondrial extracts from wt or cdc48-2 mutant cells, additionally expressing HA-Fzo1 and Cdc48, Cdc48A547T or the corresponding empty vector, as indicated, were lysed and HA-tagged Fzo1 was precipitated using HA-coupled beads. Samples were analyzed by SDS-PAGE and immunoblotting using an HA-specific antibody. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated as in Figure 1B. (C) Rescue analysis of mitochondrial morphology in cdc48-2 cells. Wt or cdc48-2 mutant cells expressing Cdc48 or Cdc48A547T or the corresponding empty vector as indicated were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid, as in Figure 1A. Quantification from three different experiments (with more than 200 cells each), including SD, as described (Cumming et al., 2007). IP, immunoprecipitation. PoS, PonceauS staining.

https://doi.org/10.7554/eLife.30015.005
Figure 2 with 1 supplement
Cdc48 specifically affects ubiquitylated Fzo1.

(A) Physical interaction between Cdc48 and ubiquitylated Fzo1. HA-Fzo1, HA-Fzo1K464R or the corresponding vector were expressed in ∆fzo1 cells. Crude mitochondrial extracts were lysed and HA-tagged Fzo1 was precipitated using HA-coupled beads and analyzed by SDS-PAGE and immunoblotting using HA- and Cdc48-specific antibodies. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated as in 1B. (B) Effect of the anti-fusion ubiquitylation of Fzo1 on its interaction with Cdc48. HA-Fzo1 or HA-Fzo1K464R, expressed in the presence of Ubp2 (∆fzo1 cells plus empty vector) or Ubp2C745S (∆ubp2 ∆fzo1 cells plus Ubp2C745S-Flag), or the corresponding vector control (the empty vectors corresponding to HA-Fzo1 and Ubp2C745S-Flag, expressed in ∆ubp2 ∆fzo1 cells), were analyzed for Cdc48 interaction, as in 2A. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated by a black arrowhead or black arrows, respectively. Red arrows with no fill indicate Fzo1 ubiquitylated species specifically accumulating upon expression of Ubp2C745S. PoS, PonceauS staining; IP, immunoprecipitation; WB, western blot.

https://doi.org/10.7554/eLife.30015.006
Figure 2—figure supplement 1
Cdc48 specifically affects ubiquitylated Fzo1.

Steady state levels of HA-Fzo1K464R upon mutation of CDC48. Total cellular extracts of ∆fzo1 or ∆fzo1 cdc48-2 mutant cells expressing HA-Fzo1 or HA-Fzo1K464R were analyzed by SDS-PAGE and immunoblotting using Fzo1-,Ubc6- and Tom40-specific antibodies. Bottom panel, quantification of four independent experiments, including SD. PoS, Ponceau S staining.

https://doi.org/10.7554/eLife.30015.007
Figure 3 with 1 supplement
Cdc48 supports ubiquitin-dependent turnover of Ubp12.

(A) Stability of the Ubp12 protein. The turnover of Ubp12 endogenously Flag tagged (Ubp12-Flagint), in wt or cdc48-2 cells, was assessed with CHX chase, as in 1D. Samples were analyzed by SDS-PAGE and immunoblotting using a Flag-, Tom40- and, as an unstable protein control, a Ubc6-specific antibody. Bottom panel, quantification of three independent experiments, including SD. (B) Ubiquitylation of Ubp12. The Ubp12C372S-Flag inactive variant, expressed from an episomal plasmid, was immunoprecipitated from total soluble extracts using Flag-coupled beads. After elution, Ubp12 was analyzed by western blot using Flag- or ubiquitin (Ub - P4D1)-specific antibodies. Ubiquitylated forms of Ubp12C372S-Flag are labeled with Ub. (C) Physical interaction between Cdc48 and Ubp12. The catalytically inactive Ubp12C372S-Flag variant, expressed from an episomal plasmid, or the corresponding empty vector, were expressed in Δubp12 (CDC48) or Δubp12 cdc48-2 (cdc48-2) mutant cells and analyzed for Cdc48 interaction. Crude mitochondrial extracts were lysed, Flag-tagged Ubp12 was precipitated using Flag-coupled beads, and the eluate analyzed by SDS-PAGE and immunoblotting using Flag- and Cdc48-specific antibodies. PoS, Ponceau S staining; IP, immunoprecipitation; WB, western blot.

https://doi.org/10.7554/eLife.30015.008
Figure 3—figure supplement 1
Cdc48 supports ubiquitin-dependent turnover of Ubp12.

(A) Turnover of episomal Ubp12 in wt or cdc48-2 cells. Ubp12-Flag stability was assessed after inhibition of cytosolic protein synthesis with cycloheximide (CHX), for the indicated time points in exponentially growing cultures. Samples were analyzed by SDS-PAGE and immunoblotting using Flag-, Ubc6- and Tom40-specific antibodies. Bottom panel, quantification of three independent experiments, including SD. (B) Proteasome dependence of Ubp12-Flag degradation. The turnover of Ubp2-Flag, expressed from an episomal plasmid, was assessed as in 1D. Samples were analyzed by SDS-PAGE and immunoblotting using Flag-, Ubc6- and Ssc1-specific antibodies. (C) Ubp12 expression levels. Expression levels of endogenously Flag-tagged Ubp12 (Ubp12-Flagint), Ubp12-Flag expressed from an episomal plasmid and endogenously Flag-tagged Ubp12 under the control of a pGAL promoter (pGAL-Ubp12-Flagint) (grown in glucose or galactose as indicated) were analyzed by SDS-PAGE and immunoblotting using Flag- and Ssc1-specific antibodies. Pos, PonceauS staining.

https://doi.org/10.7554/eLife.30015.009
Figure 4 with 2 supplements
Interdependence of Cdc48 and Ubp12 for Fzo1 regulation.

(A) Mitochondrial morphology upon deletion of UBP12 and/or mutation of CDC48. The indicated mutant cells were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid, as in Figure 1A. Right panel, quantification from three different experiments (with more than 200 cells each), including SD, as described (Cumming et al., 2007) (B) Respiratory capacity of cells upon deletion of UBP12 and/or mutation of CDC48. Fivefold serial dilutions of exponentially growing cells of wt or the mutant strains Δubp12, cdc48-2, and Δubp12 cdc48-2 were spotted on YP media supplemented with lactate (YPLac) and incubated at 30°C for two days or 37°C for five days. (C) Ubiquitylation levels of Fzo1 upon deletion of UBP12 and/or mutation of CDC48. Crude mitochondrial extracts from the indicated strains additionally expressing HA-Fzo1, or the corresponding empty vector, were analyzed by SDS-PAGE and immunoblotting using an HA-specific antibody. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated as in Figure 1B. Bottom panel, quantification of four independent experiments, normalized to PoS and including SD. ns, p>0.05. *, p≤0.05, **, p≤0.01 (One-way ANOVA, Tukey’s multiple comparison test). PoS, PonceauS staining.

https://doi.org/10.7554/eLife.30015.010
Figure 4—figure supplement 1
Interdependence of Cdc48 and Ubp12 for Fzo1 regulation.

(A) Mitochondrial morphology upon deletion of UBP12 in Δfzo1 cells. The indicated mutant cells were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid, as in Figure 1A. Quantification from three different experiments (with more than 200 cells each), including SD, as described (Cumming et al., 2007). (B) Mitochondrial morphology upon expression of HA-Fzo1 in Δfzo1 Δubp12 cells. The indicated mutant cells were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid, as in Figure 1A. Quantification from three different experiments (with more than 200 cells each), including SD, as described (Cumming et al., 2007). (C) Mitochondrial morphology upon endogenous expression of HA-Fzo1 or HA-Fzo1K464R in Δubp12 cells. The indicated mutant cells were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid, as in Figure 1A. Quantification from one experiment (with more than 200 cells each). (D) Mitochondrial morphology upon deletion of UBP12 in Δfzo1 Δdnm1 cells. The indicated mutant cells were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid, as in Figure 1A. Quantification from three different experiments (with more than 200 cells each), including SD, as described (Cumming et al., 2007).

https://doi.org/10.7554/eLife.30015.011
Figure 4—figure supplement 2
Interdependence of Cdc48 und Ubp12 for Fzo1 regulation.

(A) Analysis of mtDNA content in cdc48-2 cells using RT-PCR. mtDNA content in Δfzo1, wt and cdc48-2 cells was analyzed by measuring COX3 and ACT1 (as housekeeping gene) RNA levels using RT-PCR. Quantification of six independent experiments, including SD. *p≤0.05 (paired t-test). (B) Analysis of mtDNA content in cdc48-2 cells using the Cox2 protein amount. Total cellular extracts of Δfzo1, wt and cdc48-2 cells were analyzed by SDS-PAGE and immunoblotting using Cox2- (as mtDNA marker) or Ubc6-specific antibodies. Bottom panel, quantification of five independent experiments, including SD. *p≤0.05 (paired t-test). (C) Respiratory capacity of cdc48-2 cells upon expression of wt or mutant Cdc48. A spot assay was performed as described in Figure 4B with the indicated cells but using YPLac, grown at 30°C for 1 day and at 37°C for 3 days. (D) Physical interaction between Cdc48 and Fzo1 in Δubp12 cells. HA-Fzo1 or the corresponding empty vector was expressed in wt or Δubp12 cells and analyzed for Cdc48 interaction, as in 2A. Crude mitochondrial extracts were lysed, HA tagged Fzo1 was precipitated using HA-coupled beads, and the eluate was analyzed by SDS-PAGE and immunoblotting using HA- and Cdc48-specific antibodies. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated as in Figure 1B. (E) Steady state levels of Fzo1 upon deletion of UBP2 and/or mutation of CDC48. Total cellular extracts of wt cells or Δubp12, cdc48-2 and Δubp12 cdc48-2 mutant cells were analyzed by SDS-PAGE and immunoblotting using HA-, Ubc6- and Tom40-specific antibodies. Bottom panel, quantification of six independent experiments, including SD. ns, p>0.05 (One-way ANOVA; Tukey’s multiple comparison test). PoS, Ponceau S staining; IP, immunoprecipitation; WB, western blot.

https://doi.org/10.7554/eLife.30015.012
Figure 5 with 2 supplements
Ubp12 modulates Ubp2 ubiquitylation and turnover.

(A) Interdependent role of Ubp2 and Ubp12 for the steady state levels of Fzo1. Total cellular extracts of wt or Δubp2, Δubp12, and Δubp2 Δubp12 mutant cells expressing HA-Fzo1 and also expressing either Ubp2-Flag or the corresponding empty vector, as indicated, were analyzed by SDS-PAGE and immunoblotting using HA- and Tom40-specific antibodies. Bottom panel, quantification of four independent experiments, including SD. (B) Turnover of endogenous Ubp2 in wt or Δubp12 cells. The turnover of endogenously 3xHA-tagged Ubp2 (Ubp2-3xHAint) was assessed as in 3A. Samples were analyzed by SDS-PAGE and immunoblotting using antibodies against HA, Ubc6 and Ssc1. Right panel, quantification of four independent experiments, including SD. For the statistical analysis of the degradation kinetics of each strain, a paired t-test was used; for the statistical analysis of the difference in steady state levels of both strains at the indicated time points (t1h, t3h) an unpaired t-test was used. ns, p>0.05; *, p≤0.05; **, p≤0.01. (C) Ubiquitylation of Ubp2. The Ubp2C745S-Flag inactive variant, expressed in wt or Δubp12 cells, was immunoprecipitated from total soluble extracts using Flag-coupled beads. Eluted Ubp2 was analyzed by western blot using Flag- or ubiquitin (Ub - P4D1)-specific antibodies. Ubiquitylated forms of Ubp2C745S-Flag are labeled with Ub. PoS, Ponceau S staining; IP, immunoprecipitation; WB, western blot.

https://doi.org/10.7554/eLife.30015.013
Figure 5—figure supplement 1
Ubp12 modulates Ubp2 ubiquitylation and turnover.

(A) Opposing roles of Ubp2 and Ubp12 for CHX resistance. A spot assay was performed, as described in Figure 4B, but on synthetic media supplemented with glucose (SCD) in the absence or presence of 0.5 µg/ml CHX and incubated at 30°C for one or five days, respectively. (B) Distinct roles of Ubp2 and Ubp12 for cellular ubiquitylation. Total cellular extracts of the indicated strains were analyzed by SDS-PAGE and immunoblotting using ubiquitin (Ub; αP4D1) and Tpi1-specific antibodies, used as loading control. Free ubiquitin or ubiquitylated conjugates are labeled with Ub. Right panels, quantification of three independent experiments showing the levels of free Ub or Ub conjugates, including SD.

https://doi.org/10.7554/eLife.30015.014
Figure 5—figure supplement 2
Ubp12 modulates Ubp2 ubiquitylation and turnover.

(A) Proteasome dependence of Ubp2-Flag degradation in Δpdr5 Δsnq2 mutant cells. The turnover of ectopically expressed Ubp2-Flag was assessed as in Figure 1D. Samples were analyzed by SDS-PAGE and immunoblotting using Flag- and Ubc6-specific antibodies. (B) Physical interaction between Ubp2 and Ubp12. Catalytically inactive variants ectopically expressed Ubp2C745S-Flag and non-tagged Ubp12C372S, or their corresponding empty vectors, were expressed in Δubp2 Δubp12 cells. Total soluble extracts were prepared and Ubp12C372S was precipitated using Sepharose beads in the presence or absence of a Ubp12-specific antibody, as indicated. The eluates were analyzed by SDS-PAGE and immunoblotting using Flag- and Ubp12-specific antibodies. (C) Ubiquitylation of Ubp2. The Ubp2C745S-Flag inactive variant, expressed in wt, Δubp12 and Δubp12Δmdm30 cells, was immunoprecipitated from total soluble extracts using Flag-coupled beads. Eluted Ubp2 was analyzed by western blot using antibodies specific for Flag or ubiquitin (Ub; αP4D1). Ubiquitylated forms of Ubp2C745S-Flag are labeled with Ub. PoS, PonceauS staining; IP, immunoprecipitation; WB, western blot.

https://doi.org/10.7554/eLife.30015.015
Figure 6 with 1 supplement
Characterization of the deubiquitylation reaction by Ubp12.

(A) Analysis of ubiquitin chain-type composition of Fzo1. Crude mitochondrial extracts from wt or Δubp12 mutant cells expressing HA-Fzo1, and over-expressing either wt ubiquitin (Ub) or ubiquitin with a K48R mutation (UbK48R), were solubilized, subjected to HA-immunoprecipitation and analyzed by SDS-PAGE and immunoblotting using an HA-specific antibody. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated as in 1B. (B) Ubiquitin chain-type analysis of Fzo1 upon Ubp2C745S expression. Crude mitochondrial extracts from wt or Δubp2 (expressing Ubp2C745S) cells expressing HA-Fzo1 endogenously, and overexpressing either wt ubiquitin (Ub) or UbK48R, were analyzed as in A. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated as in 2B (C) Analysis of Ubp2 ubiquitin chain composition in Δubp12 cells. Soluble extracts from Δubp12 cells expressing Ubp2C745S-Flag and different ubiquitin variants (as indicated) were prepared and Flag-tagged Ubp2C745S was precipitated using Flag-coupled beads. The eluate was analyzed by SDS-PAGE and immunoblotting using antibodies against Flag and ubiquitin (Ub; αP4D1). (D) Deubiquitylation (DUB) assay using Ub2 chains. Purified di-ubiquitin chains (Ub2) composed of either only K48- or K63-linkages were treated with the purified DUBs Ubp12, USP21 and USP2. Treated chains were analyzed by SDS-PAGE and immunoblotting using a ubiquitin-specific antibody (Ub; αP4D1). Mono-ubiquitin or di-ubiquitin chains are labeled with Ub1 or Ub2, respectively. (E) DUB assay using Ub-chains. Purified poly-ubiquitin chains (Ub-chains) composed of either only K48- or K63-linkages were treated with the purified DUBs Ubp12, USP21 or USP2. Treated chains were analyzed by SDS-PAGE and immunoblotting as in C. Ubiquitin chains were labeled as in D with the subscript value indicating the amount of ubiquitin moieties in the respective chain. (F) Ubiquitylation pattern of Fzo1. Wt cells expressing HA-Fzo1 were analyzed for Fzo1 ubiquitylation upon the expression of Myc-ubiquitin, or the respective empty vector. HA-Fzo1 was immunoprecipitated from mitochondrial extracts using HA-coupled beads. Eluted Fzo1 was split into two and samples were analyzed by SDS-PAGE and immunoblotting using HA- or Myc-specific antibodies. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated as in 1B. The composition of the additional species apparent upon co-expression of Myc-tagged ubiquitin is explained in the inset. PoS, PonceauS staining.

https://doi.org/10.7554/eLife.30015.016
Figure 6—figure supplement 1
Characterization of the deubiquitylation reaction by Ubp12.

Opposite effects of Ubp12 and Ubp2 in Fzo1 stability. The turnover of HA-Fzo1 in wt, Δubp12, Δubp2 or Δubp12 Δubp2 cells was assessed after inhibition of cytosolic protein synthesis with cycloheximide (CHX), for the indicated time points in exponentially growing cultures. Samples were analyzed by SDS-PAGE and immunoblotting using a HA- and Hsp70-specific antibodies. Left panel, quantification of three independent experiments, including SD.

https://doi.org/10.7554/eLife.30015.017
Interdependent roles of Ubp2 and Ubp12.

(A) Effect of Ubp2C745S on Fzo1K464R ubiquitylation. HA-Fzo1 or HA-Fzo1K464R were expressed in the presence of Ubp2 (∆fzo1 cells plus empty vector) or instead in the presence of Ubp2C745S (∆ubp2 ∆fzo1 plus Ubp2C745S-Flag), as indicated. Crude mitochondrial extracts were solubilized and HA-tagged Fzo1 was analyzed by SDS-PAGE and immunoblotting using an HA-specific antibody. Unmodified and ubiquitylated forms of HA-Fzo1 are indicated as in 2B. (B) Effect of UBP2 deletion on the steady state levels of Fzo1K464R. Total cellular extracts of indicated strains expressing HA-Fzo1 or HA-Fzo1K464R as indicated were analyzed by SDS-PAGE and immunoblotting using HA- and Tom40-specific antibodies. Bottom panel, quantification of five independent experiments, including SD. (C) Effect of Ubp2 and Mdm30 on the steady state levels of Fzo1. Total cellular extracts of wt, Δubp2 and Δubp2 Δmdm30 cells expressing HA-tagged Fzo1 endogenously (HA-Fzo1int) were analyzed by SDS-PAGE and immunoblotting using HA- and Tom40-specific antibodies. Bottom panel, quantification of three independent experiments, including SD. PoS, Ponceau S staining.

https://doi.org/10.7554/eLife.30015.018
Figure 8 with 1 supplement
Cdc48 regulates mitochondrial fusion via Ubp12 and Ubp2.

(A) Steady state levels of Fzo1 in Δubp2 Δubp12 upon mutation of CDC48. Total cellular extracts of wt, cdc48-2, Δubp2 Δubp12 and Δubp2 Δubp12 cdc48-2 cells were analyzed by SDS-PAGE and immunoblotting using Fzo1- and Tom40-specific antibodies. Bottom panel, quantification of five independent experiments, including SD. (B) Steady state levels of Fzo1 in Δubp2 cells upon deletion of CDC48. Total cellular extracts of wt, cdc48-2, Δubp2 and Δubp2 cdc48-2 cells were analyzed by SDS-PAGE and immunoblotting using Fzo1- and Tom40-specific antibodies. Bottom panel, quantification of five independent experiments, including SD. (C) Mitochondrial morphology of cdc48-2 cells upon overexpression of Ubp2. Wt or cdc48-2 mutant cells expressing Ubp2 or the corresponding empty vector were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid, as in Figure 1A. Quantification from three different experiments (with more than 200 cells each), including SE, as described (Cumming et al., 2007). ns, p>0.05. **p≤0.01, ***p≤0.001 (One-way ANOVA, Tukey’s multiple comparison test). (D) Role of Ubp2 overexpression on the respiratory capacity of CDC48-deficient cells. A spot assay was performed as described in Figure 4B with the indicated cells but using synthetic media supplemented with lactate (SCLac) and incubated for 4 days. PoS, Ponceau S staining.

https://doi.org/10.7554/eLife.30015.019
Figure 8—figure supplement 1
Cdc48 regulates mitochondrial fusion via Ubp12 and Ubp2.

Physical interaction between Ubp2 and Cdc48. The catalytically inactive variant Ubp2C745S-Flag or the corresponding empty vector were expressed in Δubp12 cells and analyzed for Cdc48 interaction, as in 2A. Crude mitochondrial extracts were lysed and Flag-tagged Ubp2C745S was precipitated using Flag-coupled beads. The eluate was analyzed by SDS-PAGE and immunoblotting using Flag- and Cdc48-specific antibodies. PoS, Ponceau S staining; IP, immunoprecipitation; WB, western blot.

https://doi.org/10.7554/eLife.30015.020
Synergistic regulation of mitochondrial fusion by the Cdc48 cascade.

Cdc48 supports turnover of Ubp12, stabilizing ubiquitylation on Fzo1 that promotes mitochondrial fusion (green ubiquitins). Moreover, degradation of Ubp12 stabilizes Ubp2, facilitating the removal of ubiquitin chains on Fzo1 inhibiting mitochondrial fusion (red ubiquitins). Thereby, Cdc48 activates mitochondrial fusion via Ubp12 and Ubp2. In contrast, Cdc48 impairment blocks progression of mitochondrial fusion by actively preventing Ubp12 turnover. Ubp12 then leads to a cascade of events inhibiting mitochondrial fusion: A) removal of the pro-fusion ubiquitylated forms and B) inhibition of Ubp2, consequently leading to the accumulation of the anti-fusion ubiquitylated forms. This cascade allows a synergistic effect of Cdc48, via a DUB regulatory cascade, to effectively promote or inhibit mitochondrial fusion.

https://doi.org/10.7554/eLife.30015.021

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional information
strain (Saccharomyces cerevisiae)∆fzo1PMID: 9483801Escobar_lab_stock_number: FA2
strain (S. cerevisiae)cdc48-1PMID: 21441928Escobar_lab_stock_number: FA230
strain (S. cerevisiae)cdc48-2PMID: 21441928Escobar_lab_stock_number: FA231
strain (S. cerevisiae)cdc48-3PMID: 21441928Escobar_lab_stock_number: FA232
strain (S. cerevisiae)∆ubp2PMID: 9483801Escobar_lab_stock_number: FA260
strain (S. cerevisiae)∆ubp12PMID: 9483801Escobar_lab_stock_number: FA269
strain (S. cerevisiae)∆fzo1 ∆ubp2PMID: 23317502Escobar_lab_stock_number: FA362
strain (S. cerevisiae)∆ubp2 ∆ubp12PMID: 23317502Escobar_lab_stock_number: FA382
strain (S. cerevisiae)∆ubp12 ∆mdm30this studyEscobar_lab_stock_number: FA390UBP12::kanMX4; MDM30::kanMX4;obtained by crossing
strain (S. cerevisiae)HA-Fzo1int in wtPMID: 23317502Escobar_lab_stock_number: FA407
strain (S. cerevisiae)HA-Fzo1int in ∆ubp2PMID: 23317502Escobar_lab_stock_number: FA415
strain (S. cerevisiae)HA-Fzo1int in ∆ubp2
∆mdm30
PMID: 23317502Escobar_lab_stock_number: FA427
strain (S. cerevisiae)∆fzo1 ∆ubp12this studyEscobar_lab_stock_number: FA432FZO1::kanMX4; UBP12::kanMX4;obtained by crossing
strain (S. cerevisiae)HA-Fzo1-K464Rint in wtthis studyEscobar_lab_stock_number: FA451HA-Fzo1K464R genomically integrated with NatNT2 into RS140
strain (S. cerevisiae)wt (BY4741)PMID: 9483801Escobar_lab_stock_number: RS140
strain (S. cerevisiae)cdc48-2 ∆fzo1this studyEscobar_lab_stock_number: RS430FZO1::natNT2 in FA231
strain (S. cerevisiae)cdc48-2 ∆ubp12this studyEscobar_lab_stock_number: RS466FZO1::hphNT1 in
FA231
strain (S. cerevisiae)cdc48-2 ∆ubp2 ∆ubp12this studyEscobar_lab_stock_number: RS499UBP12::natNT2; UBP2::hphNT1 in
FA231
strain (S. cerevisiae)∆doa1PMID: 9483801Escobar_lab_stock_number: RS518
strain (S. cerevisiae)∆pdr5 ∆snq2otherEscobar_lab_stock_number: RS527gift by J. Dohmen (YGA58): MATa, ADE2 his3-D200 leu2-3,112 lys2-801, trp1D63 ura3-52 PDR5::hphNT1 SNQ2::kanMX4
strain (S. cerevisiae)Ubp12-Flagint in cdc48-2this studyEscobar_lab_stock_number: RS546Ubp12-Flag genomically integrated with NatNT2 into FA231
strain (S. cerevisiae)Ubp12-Flagint in wtthis studyEscobar_lab_stock_number: RS547Ubp12-Flag genomically integrated with NatNT2 into BY4741
strain (S. cerevisiae)∆pdr5 ∆snq2this studyEscobar_lab_stock_number: RS554PDR5::NatNT2; SNQ2::hphNT1 in RS140
strain (S. cerevisiae)∆fzo1 ∆dnm1 ∆ubp12this studyEscobar_lab_stock_number: RS556UBP12::NatNT2 in TS1028
strain (S. cerevisiae)∆pdr5 ∆snq2 cdc48-2this studyEscobar_lab_stock_number: RS559PDR5::NatNT2; SNQ2::hphNT1 in FA231
strain (S. cerevisiae)cdc48-2 ∆ubp2this studyEscobar_lab_stock_number: TS686UBP2::hphNT1 in FA231
strain (S. cerevisiae)∆fzo1 ∆dnm1otherEscobar_lab_stock_number: TS1028gift by B. Westermann (SB95): FZO1::kanMX4; DNM1::kanMX4; obtained by crossing
strain (S. cerevisiae)wt (DF5)PMID: 11007476Escobar_lab_stock_number: TS1124
strain (S. cerevisiae)ufd1-2PMID: 11847109Escobar_lab_stock_number: TS1125
strain (S. cerevisiae)npl4-1PMID: 8930904Escobar_lab_stock_number: TS1126
strain (S. cerevisiae)Ubp2-9Mycint in wtthis studyEscobar_lab_stock_number: TS1134Ubp2-9Myc genomically integrated with NatNT2 into RS140
strain (S. cerevisiae)Ubp2-3HAint in wtthis studyEscobar_lab_stock_number: TS1144Ubp2-3HA genomically integrated with hphNT1 in RS140
strain (S. cerevisiae)Ubp2-3HAint in ∆ubp12this studyEscobar_lab_stock_number: TS1147Ubp2-3HA genomically integrated with hphNT1 in FA269
strain (S. cerevisiae)pGAL-Ubp12-Flagint in wtthis studyEscobar_lab_stock_number: TS1153pGAL-Ubp12-Flag genomically integratedwith kanMX4 into RS544
recombinant DNA
reagent
pRS316 (plasmid)PMID: 2659436Escobar_lab_stock_number: p8
recombinant DNA
reagent
HA-Fzo1 on pRS316
(plasmid)
PMID: 23317502Escobar_lab_stock_number: p10
recombinant DNA
reagent
HA-Fzo1-K464R on pRS316
(plasmid)
PMID: 23317502Escobar_lab_stock_number: p14
recombinant DNA
reagent
YEplac181
(plasmid)
PMID: 3073106Escobar_lab_stock_number: p58
recombinant DNA
reagent
Ubp2-Flag on
YEplac181(plasmid)
PMID: 23317502Escobar_lab_stock_number: p59
 recombinant DNA
reagent
Ubp2-C745S-Flag on
YEplac181(plasmid)
PMID: 23317502Escobar_lab_stock_number: p60
recombinant DNA
reagent
Ubp12-Flag on
YEplac181(plasmid)
PMID: 23317502Escobar_lab_stock_number: p61
recombinant DNA
reagent
Ubp12-C372S-Flag on
YEplac181(plasmid)
PMID: 23317502Escobar_lab_stock_number: p62
recombinant DNA
reagent
YEplac195
(plasmid)
PMID: 3073106Escobar_lab_stock_number: p63
recombinant DNA
reagent
Ubp12C372S on YEplac195
(plasmid)
this studyEscobar_lab_stock_number: p65Ubp12C372S (non-tagged) on YEplac195, 2µ, Ura3
recombinant DNA
reagent
mt-GFP on pYX142
(plasmid)
PMID: 11054823Escobar_lab_stock_number: p70
recombinant DNA
reagent
Cdc48 wt on pRS313
(plasmid)
PMID: 22580068Escobar_lab_stock_number: p75
recombinant DNA
reagent
pRS313 (plasmid)PMID: 2659436Escobar_lab_stock_number: p79
recombinant DNA
reagent
Cdc48-A547T on
pRS313 (plasmid)
this studyEscobar_lab_stock_number: p150Cdc48A547T on pRS313, cen, His3
recombinant DNA
reagent
Ub on pKT10
(plasmid)
PMID: 2164637Escobar_lab_stock_number: p341
recombinant DNA
reagent
Ub-K48R on pKT10
(plasmid)
PMID: 2164637Escobar_lab_stock_number: p342
recombinant DNA
reagent
Ub-K63R on pKT10
(plasmid)
PMID: 2164637Escobar_lab_stock_number: p343
recombinant DNA
reagent
Ub-K48R,K63R on pKT10
(plasmid)
PMID: 2164637Escobar_lab_stock_number: p344
recombinant DNA
reagent
Myc-Ub on pRS426
(plasmid)
PMID: 25620559Escobar_lab_stock_number: p356
recombinant DNA
reagent
pRS426 (plasmid)PMID: 25620559Escobar_lab_stock_number: p375
Antibodyanti-Cdc48othergift by T. Sommer; (1:1,000/1:10,000)
Antibodyanti-Cox2othergift by W. Neupert; (1:5,000)
Antibodyanti-Flag M2SigmaSigma: F1804(1:1,000)
Antibodyanti-Fzo1this studyProduced by GenScript using the peptide CHGDRKPDDDPYSSS; (1:1,000)
Antibodyanti-HARocheRoche: 11867423001(1:1,000)
Antibodyanti-MycCell SignalingCell_Signaling: #2276(1:1,000)
Antibodyanti-Sec61othergift by T. Sommer; (1:10,000)
Antibodyanti-Ssc1Fölsch et al., 1998(1:40,000)
Antibodyanti-Tom40othergift by W. Neupert; (1:40,000)
Antibodyanti-Tpi1othergift by J. Dohmen; (1:5,000)
Antibodyanti-Ub (P4D1)Cell SignalingCell_Signaling: #3936(1:1,000)
Antibodyanti-Ubc6othergift by T. Sommer; (1:10,000)
Antibodyanti-Ubp12this study(1:200)
softwareMicrosoft Office 2010Micosoft
Corporation
softwareAdobe Photoshop CS6Adobe
softwareAdobe Illustrator CS6Adobe
softwareClone ManagerSci-Ed Software
softwareImage QuantGE Healthcare Life Sciences
softwareAxiovisionZeiss
softwareStepOne SystemThermo Fisher
Scientific
kitNucleoSpin RNAMachery NagelREF:740955
kitSuperScript III First-Strand Synthesis SystemInvitrogenCatalogue_number:18080051
Table 1
Yeast strains used in this study.
https://doi.org/10.7554/eLife.30015.022
Strain #Strain nameGenotypeReference
FA2fzo1FZO1::kanMX4 in BY4741Brachmann et al., 1998
FA230cdc48-1cdc48-1::KanMX4 in BY4741Li et al. (2011)
FA231cdc48-2cdc48-2::KanMX4 in BY4741Li et al. (2011)
FA232cdc48-3cdc48-3::KanMX4 in BY4741Li et al. (2011)
FA260ubp2UBP2::kanMX4 in BY4741Brachmann et al., 1998
FA269ubp12UBP12::kanMX4 in BY4741Brachmann et al., 1998
FA362fzo1ubp2FZO1::kanMX4; UBP2::kanMX4; obtained by crossingAnton et al. (2013)
FA382ubp2ubp12UBP12::kanMX4; UBP2::kanMX4; obtained by crossingAnton et al. (2013)
FA390ubp12 ∆mdm30UBP12::kanMX4; MDM30::kanMX4; obtained by crossingthis study
FA407HA-Fzo1int in wtHA-Fzo1 genomically integrated with NatNT2 into RS140Anton et al. (2013)
FA415HA-Fzo1int in ∆ubp2HA-Fzo1 genomically integrated with NatNT2 into FA260Anton et al. (2013)
FA427HA-Fzo1int in ∆ubp2mdm30HA-Fzo1 genomically integrated with NatNT2 into ∆ubp2mdm30Anton et al. (2013)
FA432∆fzo1 ∆ubp12FZO1::kanMX4; UBP12::kanMX4; obtained by crossingthis study
FA451HA-Fzo1-K464Rint in wtHA-Fzo1K464R genomically integrated with NatNT2 into RS140this study
RS140wtBY4741; S288C isogenic yeast strain; MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0Brachmann et al., 1998
RS430cdc48-2fzo1FZO1::natNT2 in FA231this study
RS466cdc48-2ubp12FZO1::hphNT1 in FA231this study
RS499cdc48-2ubp2ubp12UBP12::natNT2; UBP2::hphNT1 in FA231this study
RS518doa1DOA1::kanMX4 in BY4741Brachmann et al., 1998
RS527pdr5snq2MATa, ADE2 his3-D200 leu2-3,112 lys2-801, trp1D63 ura3-52 PDR5::hphNT1 SNQ2::kanMX4J. Dohmen (YGA58)
RS546Ubp12-Flagint in cdc48-2Ubp12-Flag genomically integrated with NatNT2 into FA231this study
RS547Ubp12-Flagint in wtUbp12-Flag genomically integrated with NatNT2 into BY4741this study
RS554pdr5snq2PDR5::NatNT2; SNQ2::hphNT1 in RS140this study
RS556∆fzo1 ∆dnm1 ∆ubp12UBP12::NatNT2 in TS1029this study
RS559pdr5snq2 cdc48-2PDR5::NatNT2; SNQ2::hphNT1 in FA231this study
TS686cdc48-2ubp2UBP2::hphNT1 in FA231this study
TS1029∆fzo1 ∆dnm1FZO1::kanMX4; DNM1::kanMX4; Mat α, BY background, obtained by crossingB. Westermann (#94)
TS1124wt (DF5)MATα, trp1-1(am), ura3-52, his3∆200, leu2-3, lys2-801Hoppe et al. (2000)
TS1125ufd1-2ufd1-2ts in TS1124Braun et al. (2002)
TS1126npl4-1npl4-1ts in TS1124DeHoratius and Silver (1996)
TS1134Ubp2-9Mycint in wtUbp2-9Myc genomically integrated with NatNT2 into RS140this study
TS1144Ubp2-3HAint in wtUbp2-3HA genomically integrated with hphNT1 in RS140this study
TS1147Ubp2-3HAint in ∆ubp12Ubp2-3HA genomically integrated with hphNT1 in FA269this study
TS1153pGAL-Ubp12-Flagint in wtpGAL-Ubp12-Flag genomically integrated with kanMX4 into RS544this study
Table 2
Plasmids used in this study.
https://doi.org/10.7554/eLife.30015.023
Plasmid #Plasmid nameDescriptionBacterial selectionReference
8pRS316pRS316, cen, Ura3AmpSikorski and Hieter, 1989
10HA-Fzo1 on pRS316HA-Fzo1 on pRS316, Fzo1 prom, cen, Ura3AmpAnton et al. (2013)
14HA-Fzo1-K464R on pRS316HA-Fzo1K464R on pRS316, Fzo1 prom, cen, Ura3AmpAnton et al. (2013)
58YEplac181YEplac181, 2µ, Leu2AmpGietz and Sugino, 1988
59Ubp2-Flag on YEplac181Ubp2-Flag on YEplac181, Adh1 prom, 2µ, Leu2AmpAnton et al. (2013)
60Ubp2-C745S-Flag on YEplac181Ubp2C745S-Flag on YEplac181, Adh1 prom, 2µ, Leu2AmpAnton et al. (2013)
61Ubp12-Flag on YEplac181Ubp2-Flag on YEplac181, Adh1 prom, 2µ, Leu2AmpAnton et al. (2013)
62Ubp12-C372S-Flag on
YEplac181
Ubp2C372S-Flag on YEplac181, Adh1 prom, 2µ, Leu2AmpAnton et al. (2013)
63YEplac195YEplac195, 2µ, Ura3AmpGietz and Sugino, 1988
65Ubp12C372S on YEplac195Ubp12C372S (non-tagged) on YEplac195, 2µ, Ura3Ampthis study
70mt-GFP on pYX142mt-GFP on pYX142, cen, Leu2AmpWestermann and Neupert, 2000
75Cdc48 wt on pRS313Cdc48 wt on pRS313, cen, His3AmpEsaki and Ogura (2012)
79pRS313pRS313, cen, His3AmpSikorski and Hieter, 1989
150Cdc48-A547T on pRS313Cdc48A547T on pRS313, cen, His3Ampthis study
341Ub on pKT10Ub on pK10, 2µ, Ura3AmpTanaka et al., 1990
342Ub-K48R on pKT10UbK48R on pK10, 2µ, Ura3AmpTanaka et al., 1990
343Ub-K63R on pKT10UbK63R on pK10, 2µ, Ura3AmpTanaka et al., 1990
344Ub-K48R,K63R on pKT10UbK48R,K63R on pK10, 2µ, Ura3AmpTanaka et al., 1990
356Myc-Ub on pRS426pCup1-Myc-Ub on pRS426, 2µ, Ura3AmpLi et al., 2015
375pRS426pRS426, 2µ, Ura3AmpLi et al., 2015
Table 3
Antibodies used in this study.
https://doi.org/10.7554/eLife.30015.024
NameDilutionReference
Cdc481:1000/1:10,000T. Sommer
Cox21:5000W. Neupert
Flag M21:1000Sigma (F1804)
Fzo11:1000this study
HA1:1000Roche (11867423001)
Myc1:1000Cell Signaling (#2276)
Sec611:10,000T. Sommer
Ssc11:40,000Fölsch et al., 1998
Tom401:40,000W. Neupert
Tpi11:5000J. Dohmen
Ub (P4D1)1:1000Cell Signaling (#3936)
Ubc61:10,000T. Sommer
Ubp121:200this study

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)