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. 2019 Jun 7;8:e45506. doi: 10.7554/eLife.45506

Figure 4. Msp1 overexpression clears mitochondrial accumulated Pex15Δ30 in doa10Δ.

(A) Flow cytometry GFP measurements of tFT-Pex15Δ30 expressed in wt, DOA10, CUE1, UBC6 and MSP1 mutant yeast (n = 4, ± SEM). Msp1 protein expression is controlled from the galactose-inducible GAL1 promoter (GAL1pr-MSP1). (B) Localization of tFT-Pex15Δ30 in strains of (A) with and without Msp1 overexpression. Scale bar: 5 μm. (C) Ubiquitylation of TAP-Pex15Δ30 in wt and doa10Δ strains expressing 10xhistidine-tagged ubiquitin as assessed by Ni-NTA affinity purification and western blotting (WB). neg. refers to a strain expressing TAP-Pex15Δ30 but not 10xhistidine-tagged ubiquitin. Tub2 is used as loading control.

Figure 4.

Figure 4—figure supplement 1. Overexpression of Msp1 restores cellular Pex15Δ30 level.

Figure 4—figure supplement 1.

(A) Lysate of yeast strains expressing 3HA-tagged Msp1 from endogenous (MSP1-3HA) and GAL1-inducible promoter (GAL1pr-MSP1-3HA) cultured in Msp1 expression restrictive (glucose) and permissive (galactose) conditions. Expression of Msp1-3HA was induced for 4 hr. Pgk1 is used as loading control. (B) Localization of Msp1-mNeonGreen under the control of GAL1 promoter. Msp1 expression was induced for 4 hr prior imaging. Mitochondrial structures are visualized by Cox4-mScarlet-i expression, magenta. Colocalization of Msp1 and Cox4 is highlighted in white in merge. Scale bar: 5 μm. (C) Cycloheximide chase of tFT-Pex15Δ30 expressing wt and doa10Δ strains overexpressing Msp1 from the GAL1-inducible promoter cultured in culture medium supplemented with galactose and quantification (n = 3, ± SEM). For comparison of tFT-Pex15Δ30 level with endogenous Msp1 expression and doa10Δ quantification from cycloheximide chases of Figure 3—figure supplement 1C is projected with dashed lines. (D) Microscopy analysis of CUE1 deficient strains expressing tFT-Pex15Δ30 in yeast in combination with msp1Δ and Msp1 overexpression for 4 hr (GAL1pr-MSP1). Scale bar: 5 μm. (E) Microscopy analysis of UBC6 deficient strains expressing tFT-Pex15Δ30 in combination with Δmsp1 and Msp1 overexpression for 4 hr similar as in (D). (F) Immunoprecipitation of tFT-Pex15Δ30 from indicated strains. Membrane was probed against GFP and ubiquitin in order to determine the ubiquitination status of the tFT-Pex15Δ30. Of note, degradation resistant lower molecular weight tFT intermediates (Khmelinskii et al., 2016) were not detected on the western blot shown (see Figure 2B for details).