Rmd9p controls the processing/stability of mitochondrial mRNAs and its overexpression compensates for a partial deficiency of oxa1p in Saccharomyces cerevisiae

Abstract

Oxa1p is a key component of the general membrane insertion machinery of eukaryotic respiratory complex subunits encoded by the mitochondrial genome. In this study, we have generated a respiratory-deficient mutant, oxa1-E65G-F229S, that contains two substitutions in the predicted intermembrane space domain of Oxa1p. The respiratory deficiency due to this mutation is compensated for by overexpressing RMD9. We show that Rmd9p is an extrinsic membrane protein facing the matrix side of the mitochondrial inner membrane. Its deletion leads to a pleiotropic effect on respiratory complex biogenesis. The steady-state level of all the mitochondrial mRNAs encoding respiratory complex subunits is strongly reduced in the Deltarmd9 mutant, and there is a slight decrease in the accumulation of two RNAs encoding components of the small subunit of the mitochondrial ribosome. Overexpressing RMD9 leads to an increase in the steady-state level of mitochondrial RNAs, and we discuss how this increase could suppress the oxa1 mutations and compensate for the membrane insertion defect of the subunits encoded by these mRNAs.

Figure 1.—

The oxa1-E65G-F229S mutant displays a thermosensitive respiratory defect. (A) A putative topology of Oxa1p is presented with the five transmembrane segments within the inner membrane of mitochondria (IM). Each circle corresponds to an amino acid, starting from residue 43, as the Oxa1p presequence is expected to be cleaved at this position (Herrmann et al. 1997). Residues altered in the oxa1 mutants (E65G, WW128AA, F229S, L240S, and K332stop) are solid circles. Residues absent in the deletion mutant oxa1-ΔL1 are shaded circles. (B) Dilution series of wild-type (WT) and oxa1-E65G-F229S mutant cells were spotted onto nonfermentable ethanol/glycerol medium and incubated for 3 days at 28° or 36°. (C) The cytochrome absorption spectra of wild-type and oxa1-E65G-F229S mutant cells, grown for 3 days on galactose medium at 36°, were recorded (see materials and methods). Absorption maxima are indicated by arrows.

Figure 2.—

Restoration of respiratory complexes assembly by overexpressing RMD9 in the oxa1-E65G-F229S mutant. (A) The arrows represent the direction of transcription of the different ORFs in the region of chromosome VII carrying the suppressor gene. The genomic fragments inserted in the plasmids are indicated by thin straight lines. YepSU27 and YepSU28 were obtained after transformation of the oxa1-E65G-F229S mutant with the high-copy library in pFL44L (see materials and methods). The plasmid YepSU29 carrying only RMD9 was obtained after PCR amplification from YepSU27 and cloning into pFL44L. The mutant cells were transformed with these different plasmids and the respiratory growth of the transformants was tested. +, growth on nonfermentable substrate. (B) The oxa1-E65G-F229S mutant was transformed with three different high-copy plasmids: YepSU27 carrying RMD9 (RMD9: RMD9 overexpression), the empty control vector pFL44L, and YepNB6 carrying OXA1 (OXA1). The transformants were patched onto minimal glucose medium lacking uracil (glucose) and replica plated on nonfermentable medium (ethanol/glycerol). The plates were incubated for 4 days at 36°. (C) Cytochrome absorption spectra of the oxa1-E65G-F229S cells transformed with the high-copy plasmids YepNB6 (OXA1) or YepSU27 (RMD9), grown on ethanol/glycerol medium for 2 (OXA1) and 4 (RMD9) days at 36°, were recorded as described in the legend of Figure 1.

Figure 3.—

Defect in respiratory complex assembly in the Δrmd9 mutant. (A) Dilutions of wild-type (WT) and Δrmd9 strains were spotted onto glucose and onto glycerol media. Photographs were taken after 1 day (glucose) and 3 days (glycerol) of incubation at 28°. (B) Cytochrome absorption spectra of the wild-type and Δrmd9 cells grown on galactose medium (see legend Figure 1). (C) Mitochondria from wild-type and Δrmd9 strains, grown on galactose medium, were analyzed by SDS–PAGE (12%) and immunoblotting with anti-Cox2p, anti-Cytbp, anti-Atp6p, and anti-Cyt1p antibodies.

Figure 4.—

Mitochondrial sublocalization of Rmd9p and Ybr238p. Mitochondria were purified from galactose-grown cells expressing Rmd9p-c-Myc or Ybr238p-HA (see material and methods). Proteins were analyzed by SDS–PAGE and immunoblotting with anti-c-Myc, anti-HA, and control antibodies recognizing the soluble mitochondrial matrix protein Arg8p, the cytosolic protein PGKp, the integral mitochondrial membrane protein Cox2p, and the intermembrane space protein Cytb2p. (A) Mitochondrial (M) and postmitochondrial supernatant (S) protein fractions were separated on SDS–PAGE (12%). (B) Mitochondria were alkali treated at pH 11.5 and centrifuged at 50,000 rpm for 1 hr, according to Lemaire et al. (2000), to separate the soluble proteins recovered in the supernatant (S) from the integral membrane proteins that remained in the pellet (P). Proteins were separated on SDS–PAGE (10%). Note that the anti-HA antibody reveals a doublet on 10% SDS–polyacrylamide gels. (C) Mitochondrial proteins extracted from Rmd9p-c-Myc cells (lane 1) were treated with 100 μg/μl of proteinase K before (lane 2) or after swelling (lane 3) to break the outer membrane while keeping the inner membrane intact. The different samples were analyzed by SDS–PAGE (10%).

Figure 5.—

Analysis of mitochondrial transcripts in the Δrmd9 mutant. Total RNAs were isolated from wild-type (WT, CW252) and Δrmd9 strains grown in galactose medium at 28°. Isolation of total RNAs was repeated twice and each preparation was analyzed on several blots using the 0.5- to 10-kb RNA ladder as a size marker (Invitrogen). Equivalent amounts of 2 and 6 μg were analyzed by hybridization with various mitochondrial probes: (A) Genes encoding the mitoribosomal protein, Var1p, the two rRNAs 21S and 15S, the tRNA^glu, and Cyt1p as control. For the small tRNA^glu, we checked that it was absent in the control rho° strain. (B) Genes encoding respiratory complex subunits: CYTB, COX2, COX3, COX1, ATP6/8, ATP9, and the 21S rRNA as control. The positions of size markers are indicated at the right of the gel. (C) Schematic of the transcript units analyzed in A and B. ATP9 and VAR1, tRNA^glu, and CYTB as well as COX1 and ATP6/8 are cotranscribed.

Figure 6.—

Effect of the overexpression of RMD9 on the accumulation of mitochondrial transcripts. The wild-type (CW252) and the oxa1-E65G-F229S strains were transformed with the empty control vector pFL44L (−) and YepSU27 carrying RMD9 (RMD9). Total RNAs were isolated from the transformants grown in minimal medium to maintain the plasmids. (A) RNAs (2 μg) were analyzed by hybridization with probes specific for the nuclear gene RMD9, for the protein-encoding mitochondrial genes COX2 and ATP9, as well as for the mitochondrial rRNAs 21S and 15S (see materials and methods). (B) mRNA levels were quantified with ImageQuant (Molecular Dynamics, Sunnyvale, CA) and normalized to the levels of actin mRNA (ACT1). The histogram presents the steady-state level of three representative mitochondrial RNAs (ATP9, COX2, and 15S) in the strain overexpressing RMD9 as compared to their level in the strain transformed with the control vector.