Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins
- Centre for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Germany
- Institute of Molecular Biology (IMB), Germany
- Calico Life Sciences LLC, United States
- German Cancer Research Center (DKFZ), Germany
Figures
Figure 1 with 1 supplement
Genome-wide screen to identify factors stabilizing Pex15Δ30 TA protein.
(A) Overview of the tFT-Pex15Δ30 and tFT-Tom5 TA protein reporters. Tail-anchor is highlighted in blue. Strains, expressing either tFT-Pex15Δ30 or tFT-Tom5 from the TEF1 promoter were crossed into …
Figure 1—figure supplement 1
Colocalization of Pex15Δ30 reporter with cellular markers.
(A) Colocalization studies of wt yeast expressing GFP-Pex15Δ30 (green) with markers for mitochondria (Cox4-mScarlet-i, magenta), peroxisomes (Pex3-mScarlet-i, magenta) and ER (Sec63-mScarlet-i, …
Figure 2 with 1 supplement
ER insertion and mitochondrial accumulation impedes efficient Pex15Δ30 degradation in spf1Δ and get3Δ.
(A) Microscopy analysis of spf1Δ strains expressing GFP-Pex15Δ30 from the TEF1 promoter. Co-expression of chromosomally tagged cellular marker proteins: Cox4-mScarlet-i, mitochondria; …
Figure 2—figure supplement 1
Steady state level of tFT-Pex15Δ30 in yeast mutants.
Lysate of wt yeast and indicated mutants probed with anti-GFP antibody to reveal full length tFT-Pex15Δ30 (FL) and degradation resistant tFT intermediates (h, p, v, see Figure 2B for further …
Figure 3 with 1 supplement
Doa10 triggers degradation of cytoplasmic Pex15Δ30.
(A) Flow cytometry measurements of wt, msp1Δ, doa10Δ and msp1Δdoa10Δ strains expressing tFT-Pex15Δ30. Mean GFP intensities normalized to wt (n = 4, ± SEM). (B) Western blot (WB) analysis from log …
Figure 3—figure supplement 1
Targeted screen in ubiquitin-proteasome system mutants emphasizes role of Doa10 in Pex15Δ30 turnover.
(A) sfGFP intensities and mCherry/sfGFP ratios (n = 4), normalized to the median of each screen, for tFT-Pex15Δ30 and tFT-Tom5 expressed in an array of mutants in the ubiquitin-proteasome system. (B)…
Figure 4 with 1 supplement
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 …
Figure 4—figure supplement 1
Overexpression of Msp1 restores cellular Pex15Δ30 level.
(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 …
Figure 5 with 1 supplement
Doa10 controls targeting fidelity of mitochondrial TA proteins.
(A) Mean GFP fluorescence of 55 N-terminally GFP-tagged TA proteins expressed from NOP1 promoter deleted for DOA10 or MSP1 compared to wt. Measurements were taken from colonies grown on agar (n = 4).…
Figure 5—figure supplement 1
Msp1 does not require Cis1 for Fmp32 turnover.
Steady state analysis of wt, msp1Δ and cis1Δ strains expressing sfGFP-Fmp32 from its endogenous locus and quantification (n = 3, ± SEM).
Figure 6 with 1 supplement
Protein dimerization impedes Msp1-dependent extraction.
(A) Scheme of rapamycin-induced dimerization for the two reporter proteins FRB1TMD and FKBP12TMD. (B) Microscopy analysis of wt yeast expressing FRB1TMD and FKBP12TMD. Images were taken with and …
Figure 6—figure supplement 1
Enhanced membrane association impedes Msp1-dependent extraction of Pex15-derived reporters from mitochondria.
(A) Hydrophobicity blots of Msp1 substrates Pex15, Fmp32 and Gem1 calculated using ProtParam (Gasteiger et al., 2005). TM region (TMD) and putative juxtamembrane hydrophobic patch are indicated. …
Figure 7
Model of Doa10-mediated TA protein abundance control increasing targeting fidelity and removing clients of Msp1 dislocase.
TA proteins are post-translationally targeted to the ER by the GET pathway or insert into the OMM by an so far unknown mechanism. In the OMM, TA proteins become subject for Msp1-mediated extraction …
Additional files
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Supplementary file 1
sfGFP and mCherry/sfGFP ratio z-scores measured for tFT-Pex15Δ30 and tFT-Tom5 in the non-essential yeast deletion collection.
- https://doi.org/10.7554/eLife.45506.015
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Supplementary file 2
Array of investigated TA proteins according to Burri and Lithgow (2004).
- https://doi.org/10.7554/eLife.45506.016
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Supplementary file 3
Yeast strains used in this study.
- https://doi.org/10.7554/eLife.45506.017
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Supplementary file 4
Plasmids used in this study.
- https://doi.org/10.7554/eLife.45506.018
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Transparent reporting form
- https://doi.org/10.7554/eLife.45506.019
