Extended Data Fig. 6: Mge1’s presequence is required for inducing the mitoCPR.
From: A direct role for a mitochondrial targeting sequence in signalling stress
(a) Immunoblot of overexpressed mCherry-tagged Mge1 truncation mutants and the Mss116’s MTS (4 h following galactose induction). OE, overexpression. (b) Live-cell fluorescence images of cells expressing the nuclear envelope marker Nup159-GFP and ΔMTS-Mge1-mCherry or NLS-ΔMTS-Mge1-mCherry. Expression of MGE1 variants from the GAL1-10 promoter was induced by the addition of galactose for 4 h. Scale bars, 5 μm. (c) Pdr3-dependent upregulated genes were identified by differential gene expression analysis of: 1. wild-type versus pdr3Δ cells, both overexpressing MTSMge1-mCherry, and 2. wild-type versus pdr3Δ cells, both overexpressing PSD1. The scatter plot displays genes with an adjusted p value ≤ 0.05 (Wald test in DESeq2) in at least one of analyses. PDR3 was removed from both datasets prior to the correlation analysis. (d) Growth curves of wild-type, MTSSu9-MGE1, and MTSIlv2-MGE1 cells were plotted on a semilogarithmic scale. Corresponding growth rate (r) was calculated from the growth curves of three biological replicates, each with three technical replicates; two-tailed Student’s t-test. ns, not significant. (e) Immunoblot of cells expressing Mge1-FLAG, MTSSu9-Mge1-FLAG, or MTSIlv2-Mge1-FLAG under control and PSD1 overexpression conditions. p1, Mge1-FLAG precursor; p2, MTSSu9-Mge1-FLAG precursor; p3, MTSIlv2-Mge1-FLAG precursor. m1/2/3, mature form Mge1-FLAG/MTSSu9-Mge1-FLAG/MTSIlv2-Mge1-FLAG (f) V5-Pdr3 was immunoprecipitated using V5-trap beads from the following: 1. cells under basal conditions (n = 2 biological repeats), 2. cells under impaired mitochondrial protein import conditions (PSD1 overexpression; n = 2 biological repeats), or 3. MTSIlv2-MGE1 cells under impaired mitochondrial protein import conditions (n = 3 biological repeats). Mge1 intensities were measured by mass spectrometry analysis. Data are shown as mean; one-way ANOVA followed by Dunnett’s test. (g) Left- Confirmation of PSD1 overexpression by qPCR analysis for the samples used in Fig. 4e. n = 3 biological replicates; one-way ANOVA followed by Tukey’s test. Right- Immunoblot of Mge1-FLAG and MTSIlv2-Mge1-FLAG under control or import stress (PSD1 overexpression for 4 h) conditions. Addbacks of Mge1-mCherry and Mge1-NES-FLAG into the MTSIlv2-MGE1 background are also presented. p1, Mge1-FLAG precursor; p2, MTSIlv2-Mge1-FLAG precursor; p3, Mge1-NES-FLAG precursor; p4, Mge1-mCherry precursor; m1/2, mature Mge1-FLAG/MTSIlv2-Mge1-FLAG; m3, mature Mge1-NES-FLAG; m4, mature Mge1-mCherry. OE, overexpression. (h) Left- Confirmation of PSD1 overexpression by qPCR analysis for the samples used in Fig. 4f. n = 3 biological replicates; one-way ANOVA followed by Tukey’s test. Right- Immunoblot of Mge1-FLAG and MTSSu9-Mge1-FLAG under control or import stress (PSD1 overexpression for 4 h) conditions. Addback of Mge1-mCherry into the MTSSu9-MGE1 background is also presented. p1, Mge1-FLAG precursor; p2, MTSSu9-Mge1-FLAG precursor; p3, Mge1-mCherry precursor; m1/2, mature Mge1-FLAG/MTSSu9-Mge1-FLAG; m3, mature Mge1-mCherry. OE, overexpression. (i) CIS1 and PSD1 mRNA levels in cell expressing MTSIlv2-MGE1-FLAG and MGE1-FRB with or without the plasma membrane (PM) anchor Pil1-FKBP. Cells were grown under control or import stress (PSD1 overexpression for 4 h) conditions. Anchoring of the Mge1 precursor was induced by adding 50 nM rapamycin for the duration of PSD1 overexpression. n = 3 biological replicates; one-way ANOVA followed by Tukey’s test. (j) Immunoblot of Mge1-FLAG and MTSIlv2-Mge1-FLAG in wild-type cells, rho− cells, tam41∆ cells (at both 30 °C and 37 °C), and cells expressing aac2A128P, A137D for 4 h. p1, Mge1-FLAG precursor; p2, MTSIlv2-Mge1-FLAG precursor; m1/2, mature form Mge1-FLAG/MTSIlv2-Mge1-FLAG. (d, g-i) Data represent mean +/− SD; ns, not significant; **** P ≤ 0.0001.
