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. 2019 Dec 19;3(1):e201900476. doi: 10.26508/lsa.201900476

Figure 2. Rescue of Fzo1 protein levels is not sufficient to restore mitochondrial fusion.

Figure 2.

(A) Stability and proteasome dependence of endogenously HA-tagged Fzo1 and Fzo1K398R. Exponentially growing cells defective for the multidrug exporter Pdr5 (Δpdr5), expressing endogenously HA-tagged Fzo1 (wt) or Fzo1K398R (K398R), were treated with proteasomal inhibitor MG132 (or DMSO as a control) for 1 h before administration of the translation inhibitor cycloheximide (CHX). Samples were taken 0, 1, or 3 h after protein synthesis was shut off with CHX. Total cellular extracts were prepared and analysed by SDS–PAGE and Western blot using HA-specific and Ubc6-specific (as an unstable protein control) antibodies. (B) Rescue of HA-Fzo1K398R steady state levels by ectopic expression of HA-Fzo1K398R. HA-Fzo1K398R (or the corresponding vector control) was ectopically expressed using a centromeric plasmid in cells already expressing genomically HA-tagged Fzo1K398R, which were grown at 30°C, 30°C with a subsequent shift to 37°C for 3 h or 37°C, as indicated. Total cellular extracts were analysed by SDS–PAGE and Western blot using an HA-specific antibody. (C) Mitochondrial morphology of cells expressing equal levels of HA-tagged Fzo1 and Fzo1K398R. (B) Mitochondrial morphology was analysed as in Fig 1B using cells as described in (B). PoS, Ponceau S.