Figure 2: Ubp2 antagonizes the degradation of Fzo1 that is mediated by Mdm30. | Nature Communications

Figure 2: Ubp2 antagonizes the degradation of Fzo1 that is mediated by Mdm30.

From: An ubiquitin-dependent balance between mitofusin turnover and fatty acids desaturation regulates mitochondrial fusion

Figure 2

(a) Quantification of CHX chases (bottom) with WT, mdm30Δ, ubp2Δ and ubp2Δ mdm30Δ cells (MCY572 background) shuffled with pRS414-FZO1-13MYC were analysed by anti-Myc and anti-Pgk1 immunoblotting followed by detection with fluorescent secondary antibodies (top). Error bars represent s.e.m. from three independent experiments. (b,c) MW in kDa are shown on the right of short and long exposures of indicated regions of anti-Myc or anti-Pgk1 immunoblots. (b) Total protein extracts prepared from strains used in a but shuffled with pRS414-TEF-FZO1-13MYC were analysed by anti-Myc immunoblotting. Loading was normalized to Fzo1-13Myc levels. (c) Total protein extracts prepared from FZO1, mdm30Δ, ubp2Δ and ubp2Δ mdm30Δ cells (MCY572 background) shuffled with pRS414-TEF-FZO1-13MYC (WT) or pRS414-TEF-FZO1-13MYC K398R were analysed by anti-Myc and anti-Pgk1 immunoblotting. (d) In vitro deubiquitylation assays. Ub5K63 or Ub5K48 chains were incubated with mock (−) or Ubp2-HA immunoprecipitates in the absence (−) or in the presence of DTT (0.1 or 10 mM) or H2O2 (0.5 mM). Reactions were analysed by anti-HA and anti-Ub immunoblotting. (e) Bottom: same as (d) but with Ub(3-7) K48 chains. Top: quantification of the level of each length of chain relative to the mock (−Ubp2) condition.

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