Figure 6. Protein dimerization impedes Msp1-dependent extraction.

(A) Scheme of rapamycin-induced dimerization for the two reporter proteins FRB1^TMD and FKBP12^TMD. (B) Microscopy analysis of wt yeast expressing FRB1^TMD and FKBP12^TMD. Images were taken with and without rapamycin treatment for the indicated time. Scale bar: 5 μm. (C) Cycloheximide chase of strains from (B) with and without rapamycin pre-treatment for 30 min. Quantification of FRB1^TMD (n = 3, ± SEM) normalized to t = 0 of untreated sample. WB, western blot. Tub2 is used as loading control. (D) Cycloheximide chase of strains expressing FRB1^TMD and cytosolic FKBP12 (FKBP12^soluble) with and without rapamycin pre-treatment for 30 min. Quantification of FRB1^TMD (n = 3, ± SEM) normalized to t = 0 of untreated sample. Unspecific bands are marked with star. Pgk1 is used as loading control. (E) Steady state analysis of sfGFP-Gem1 expressed from the NOP1 promoter in wt, msp1Δ, doa10Δ and msp1Δdoa10Δ with quantification (n = 6 ± SEM, star indicates Student’s t-test p<0.05).

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. Numbers indicate amino acid position in native proteins. (B) Alignment of membrane regions from known Msp1 clients. TM domain underlined (see Chen et al., 2014; Li et al., 2019; Okreglak and Walter, 2014 and this study). Numbers indicate amino acid position. (C) Microscopy analysis of yeast strains co-expressing FRB1^TMD and mChe-FKBP12^TMD, or mChe-FKBP12^soluble, with and without rapamycin treatment for 30 min as indicated. Scale bar: 5 μm. (D) Cycloheximide chase of strains co-expressing FRB1^TMD and mChe-FKBP12^TMD with and without rapamycin pre-treatment for 30 min with quantification of FRB1^TMD and mChe-FKBP12^TMD (n = 3, ± SEM). Tub2 serves as loading control. (E) Quantification of FKBP12^TMD from Figure 6C. (F) Steady state analysis of FRB1^TMD in wt, msp1Δ and doa10Δ strains. Pgk1 serves as loading control. Pgk1 serves as loading control. (F) Strains expressing FRB1^TMD after 30 min rapamycin treatment followed by a cycloheximide chase. Pgk1 serves as loading control. (G) Cycloheximide chase of strains co-expressing FRB1^TMD and mChe-FKBP12^soluble with and without rapamycin pre-treatment for 30 min with quantification of FRB1^TMD (n = 3, ± SEM). Tub2 serves as loading control.