Role of positively charged transmembrane segments in the insertion and assembly of mitochondrial inner-membrane proteins

Abstract

The biogenesis of membrane oligomeric complexes is an intricate process that requires the insertion and assembly of transmembrane (TM) domains into the lipid bilayer. The Oxa1p family plays a key role in this process in organelles and bacteria. Hell et al. (2001, EMBO J., 20, 1281-1288) recently have proposed that Oxa1p could act as part of a general membrane insertion machinery for mitochondrial respiratory complex subunits. We have previously shown that mutations in the TM domain of Cyt1p can partially compensate for the absence of Oxa1p. Here, we demonstrate that a single amino acid substitution in the TM domain of Qcr9p can bypass Oxa1p in yeast. Qcr9p and Cyt1p are two subunits of the respiratory complex bc1 and their relative roles in the assembly of other respiratory complexes have been investigated. The mutations we have isolated in Cyt1p or Qcr9p introduce positively charged amino acids, and we show that the mutant TM domain of Cyt1p mediates the restoration of complex assembly. We propose that the positive charges introduced in Cyt1p and Qcr9p TM domains promote interactions with negatively charged TM domains of other respiratory complex subunits, allowing the coinsertion of both domains into the membrane, in the absence of Oxa1p. This model argues in favor of a role of Oxa1p in the insertion and the lateral exit of less hydrophobic TM domains from the translocation site into the lipid bilayer.

Figure 1

Schematic representation of the mitochondrial respiratory chain. Four respiratory complexes are embedded within the inner membrane of mitochondria: the complex I, III or the bc1 complex, IV or the cytochrome c oxidase complex (Cox) and the insoluble part of complex V or ATP synthase. c represents cytochrome c. bc1 and Cox complexes each contain about 10 subunits. Cyt1p and Qcr9p are nucleus-encoded subunits of bc1 complex and have one TM domain each. Cox2p is a mitochondria encoded subunit of Cox complex with two TM domains. The small arrows indicate the electron (e) flow.

Figure 2

The QCR9–1 mutation partially compensates for the respiratory defect caused by the absence of Oxa1p. WT (CW30); Δoxa1 (NBT2); Δoxa1 CYT1–1 (R101); Δoxa1 QCR9–1 (R102). See Materials and Methods and Hamel et al. (21). (A) Strains were grown on glucose (G) and ethanol/glycerol (E) media (see Materials and Methods) and incubated for 7 days at 28°C. (B) The mitochondrial translation products were ³⁵S-labeled in vivo in the presence of cycloheximide as described (28). The proteins were separated on 16% SDS-polyacrylamide gels. Arrowheads indicate apocytochrome b (Cytbp) of bc1 complex, pre-Cox2p, and mature Cox2p of cytochrome c oxidase. (C) Mitochondria were purified from cells grown on complete galactose medium as described in Materials and Methods. One hundred micrograms of purified mitochondrial proteins was separated on 12% SDS-polyacrylamide gels, and the accumulation of Cox2p, Atp4p, and Atp2p subunits was analyzed with specific antibodies by Western blotting. Atp2p is used as an internal control as we have previously shown that the accumulation of Atp2p does not depend on Oxa1p (14).

Figure 3

Respiratory growth is severely impaired in the double mutant CYT1–1 QCR9–1. WT (CW30); CYT1–1 (PHT2); QCR9–1 (PHT28); QCR9–1 CYT1–1 (CQ2–2B). (A) Strains were grown on glucose (G) and ethanol (E) media as a respiratory substrate and incubated for 3 days at 28°C. (B) Low-temperature cytochrome absorption spectra of whole cells were recorded according to ref. . The arrows indicate the absorption maxima of the α bands of cytochromes c (546 nm), c1 (552 nm), b (558 nm), and a + a3 (602 nm).

Figure 4

Cyt1p is essential for the restoration of Cox assembly in the absence of Oxa1p. (A) Mitochondrial translation products were labeled as described in Fig. 2B. WT (CW30); Δoxa1 CYT1–1 Δqcr9 (YST3); Δoxa1 Δcyt1 QCR9–1 (YST4); Δcyt1 QCR9–1 (YSG9–3B). (B) Enzymatic activities were measured in mitochondria purified from galactose grown cells as described in Materials and Methods. The cytochrome c oxidase activity (Cox activity) is expressed in nmol of oxidized cytochrome c/min per mg of protein and percentages of wild-type activities are given in parenthesis. (C) Qcr9p was tagged with the c-myc epitope as described in Materials and Methods. Five hundred micrograms of total yeast proteins, extracted as described (31), was separated on 12% SDS-polyacrylamide gels and immunoblotted with anti-c-myc. WT (YS18–5A), QCR9–1 (YS16–1B), Δoxa1 QCR9–1 (YS16–18D), Δoxa1 Δcyt1 QCR9–1 (YS16–1C).

Figure 5

Carbonate-extractable forms of cytochrome c1 do not accumulate in the Δoxa1 QCR9–1 strain. Mitochondria (equivalent of 300 μg of proteins) were purified from cells grown on galactose complete medium and treated with sodium carbonate as described (14). The supernatants (S; soluble fractions) and pellets (P, membrane fractions) were separated on 12.5% SDS-polyacrylamide gels and transferred to nitrocellulose filters. Detection of cytochromes c and c1 was performed on the membrane as described (34). * indicates the fast-migrating extractable forms of cytochrome c1. WT (CW30); Δoxa1 QCR9–1 (R102); Δoxa1 CYT1–1 (R101).

Figure 6

The mutant membrane domain of Cyt1p mediates suppression. (A) Schematic representation of the mutant pre-Cyt1p protein and the short form (see Materials and Methods for constructions). The black box represents the hydrophobic domain anchoring the protein to the membrane, and * indicates the position of the CYT1–1 mutation. The dashed box represents the 61-aa presequence that is cleaved to give the mature form Cyt1p and the four vertical lines indicate the positions of the heme ligands within the hydrophilic central domain. (B and C) The Δoxa1 mutant (NBT2) was transformed with three plasmids. YEpYS7 expresses the mutant Cyt1p protein (Δoxa1 CYT1–1), pFL61 is the control vector (Δoxa1), and YEpYS9 expresses the short form of the mutant Cyt1p (Δoxa1 CYT1–1,-S). (B) Transformants were grown on ethanol medium and incubated for a week at 28°C. (C) Mitochondria were purified from transformants grown on minimal medium to select for the presence of the plasmid. Cox2p, Atp4p, and Atp2p were immunodetected as described in Fig. 2C.

Figure 7

Mode of action of Oxa1p and positively charged TM domains. (A) Oxa1p could allow the membrane insertion of charged or less hydrophobic TM domains (e.g., first TM helix of Cox2p) by sandwiching them until they reach stable integration within the oligomeric complex (Cox complex). (B) Positively charged TM domain (e.g., Cyt1p) could be coinserted with a negatively charged one (e.g., first TM helix of Cox2p).