Peripheral mitochondrial inner membrane protein, Mss2p, required for export of the mitochondrially coded Cox2p C tail in Saccharomyces cerevisiae

Abstract

Cytochrome oxidase subunit 2 (Cox2p) is synthesized on the matrix side of the mitochondrial inner membrane, and its N- and C-terminal domains are exported across the inner membrane by distinct mechanisms. The Saccharomyces cerevisiae nuclear gene MSS2 was previously shown to be necessary for Cox2p accumulation. We have used pulse-labeling studies and the expression of the ARG8(m) reporter at the COX2 locus in an mss2 mutant to demonstrate that Mss2p is not required for Cox2p synthesis but rather for its accumulation. Mutational inactivation of the proteolytic function of the matrix-localized Yta10p (Afg3p) AAA-protease partially stabilizes Cox2p in an mss2 mutant but does not restore assembly of cytochrome oxidase. In the absence of Mss2p, the Cox2p N terminus is exported, but Cox2p C-terminal export and assembly of Cox2p into cytochrome oxidase is blocked. Epitope-tagged Mss2p is tightly, but peripherally, associated with the inner membrane and protected by it from externally added proteases. Taken together, these data indicate that Mss2p plays a role in recognizing the Cox2p C tail in the matrix and promoting its export.

FIG. 1

Cox2p is unstable in the absence of Mss2p. (A) Cox2p steady-state levels are reduced in the mss2Δ mutant. Whole-cell extracts were prepared from cells grown overnight in YPR (see Materials and Methods). Extracts were analyzed by Western blotting with anti-Cox2p and anti-glucose-6-phosphate dehydrogenase (G6P) as a loading control. Lanes: 1, wild-type (PTH366); 2, pet111 mutant (ECS108); 3, mss2Δ mutant (SB12). (B) Cox2p is synthesized but rapidly degraded in an mss2Δ mutant. Cells were incubated with cycloheximide and either pulsed with [³⁵S]methionine for 10 min and chased with cold methionine for 50 min or pulsed for 10 min with no chase, as indicated (see Materials and Methods). Mitochondria were isolated, and translation products were separated by SDS–15% PAGE and detected by autoradiography. Lanes: 1 and 4, wild type (WT; PTH366); 2 and 5, pet111 (ECS108); 3 and 6, mss2Δ (SB12).

FIG. 2

Expression of a cox2::ARG8^m reporter gene in mtDNA is independent of Mss2p. Cells were spotted onto synthetic complete medium lacking arginine or synthetic complete medium containing arginine as indicated. Plates were incubated for 2 days at 30°C. Strains: WT, wild-type HMD22; mss2, SB20; pet111, NSG192.

FIG. 3

MSS2 function cannot be bypassed by placing the COX3 mRNA 5′-UTL on the COX2 mRNA. Haploid cells containing a synthetic ρ⁻ mtDNA bearing a chimeric gene specifying a COX2 mRNA with the 5′-UTL of the COX3 mRNA (33) were patched in horizontal stripes. Their relevant nuclear genotypes were mss2 pet111 (SB26) and MSS2 pet111 (SB23B). Cells containing wild-type ρ⁺ mtDNA were patched in vertical stripes. Their relevant nuclear genotypes were mss2 PET111 (SB12) and MSS2 pet111 (NB39-9c). The stripes were cross-printed on complete medium, and diploids were selected. The diploids were printed to nonfermentable medium (YPEG) and incubated for 2 days at 30°C.

FIG. 4

Inactivation of Yta10p stabilizes Cox2p in an mss2Δ mutant but does not restore respiratory function. (A) Cells were incubated with cycloheximide, pulsed with [³⁵S]methionine for 10 min (lanes 1 to 4), and chased with cold methionine for 50 min (lanes 5 to 8). Crude mitochondria were analyzed on an SDS–15% polyacrylamide gel, followed by autoradiography. Lanes: 1 and 4, wild type (WT; J303-1a); 2 and 6, mss2Δ (SB12); 3 and 7, yta10E559Q (YGS103); 4 and 8, mss2Δ yta10E559Q double mutant (SB49C). (B) Cells were grown overnight in complete medium and spotted onto YPD and YPEG as indicated or used to prepare whole-cell extracts. Equal amounts of extracts were analyzed by Western blotting with anti-Cox2p and anti-glucose-6-phosphate dehydrogenase (G6PD). Lanes: 1, wild type (WT; J303-1a); 2, mss2Δ (SB48B); 3, yta10E559Q (YGS103); 4, yme1Δ (SB100); 5, mss2Δ yme1Δ (SB101); 6, mss2Δ yta10EQ (SB49C); 7, mss2Δ yta10EQ yme1Δ (SB103).

FIG. 5

Mss2p is required for export of the Cox2 C terminus. (A) mss2 mutants are defective in assembly of Cox2p into the proteolytically resistant cytochrome oxidase complex. Purified mitochondria (180 μg) derived from wild type (WT; DBY947) or the mss2::Tn mutant (SB44) were solubilized in 1% octylglucopyranoside and incubated with proteinase K at the indicated concentrations. Samples were analyzed by Western blotting with a monoclonal antibody that recognizes an epitope in the Cox2p C terminus (CCO6) (21). Because steady-state levels of Cox2p are reduced in the mss2::Tn mutant, detection of Cox2p in mss2::Tn extract required a longer exposure time than detection of Cox2p in wild-type extract. (B) The Cox2p C-terminal domain is protected from protease by the inner membrane of mitoplasts. Mitochondria (180 μg) from wild type (WT; DBY947) or mss2::Tn (SB44) were incubated with or without 25 μg of proteinase K/ml or converted to mitoplasts and incubated with or without 25 μg of proteinase K/ml, as indicated. Samples were analyzed by Western blotting with the Cox2p antibody (CCO6), the cytochrome b₂ antibody, or the citrate synthase antibody. Cytochrome b₂ is a IMS marker which is used to assess mitoplasting efficiency. Citrate synthase is a matrix space marker used to demonstrate that the mitochondrial inner membrane is still intact.

FIG. 6

Mss2p is not required for export of the Cox2 N terminus. (A) The N tail of Cox2 (1-67)-Arg8^mp is exported and susceptible to exogenously added protease. Purified mitochondria and mitoplasts derived from wild type (WT; SH36) or mss2Δ (SB35) cells were incubated with or without 75 μg of proteinase K/ml, and samples were analyzed by Western blotting with anti-Arg8p, anti-b₂, and anti-citrate synthase (C.S.). (B) The leader peptide of pre-Cox2p is processed normally in the absence of Mss2p. Cells were incubated in the presence of cycloheximide and pulsed for 10 min with [³⁵S]methionine. Mitochondrial translation products were separated on an SDS–12% polyacrylamide gel and analyzed by autoradiography. (The relative mobilities of Cox2p and cytochrome b are reversed on 12% gels relative to the 15% gels used in other figures.) Strains: wild type (WT; PTH366); pet111, ECS108; mss2, SB12; imp1, SH105. The mature cleaved form of Cox2p is indicated as mCox2p, while uncleaved pre-Cox2p is indicated as pCox2p.

FIG. 7

Mss2p is a mitochondrial matrix protein that is peripherally associated with the inner membrane. (A) Whole-cell extracts derived from cells containing MSS2-HA or MSS2 were analyzed by Western blotting with the antibody against the HA epitope (3F10) and anti-Arg8p. Lanes: 1, MSS2 (TF215); 2, MSS2-HA (SB19A). (B) Mss2p-HA copurifies with mitochondria. Purified mitochondria (see Materials and Methods) (lane 1) or cytosol (lane 2) derived from the MSS2-HA strain (SB19A) were analyzed by Western blotting with anti-HA, anti-Arg8p, and anti-glucose-6-phosphate (G6PD). (C) Mss2p-HA is a peripheral membrane protein. Purified mitochondria from the MSS2-HA strain were sonicated and centrifuged to separate the insoluble membrane fraction (lane 1) and the soluble fraction (lane 2). The membrane pellet was extracted with sodium carbonate and centrifuged to separate insoluble integral membrane proteins (lane 3) from solubilized peripheral membrane proteins (lane 4). Samples were analyzed by using Western blotting with anti-HA, anti-Arg8p, and anti-Cox2p. (D) Mss2p-HA is largely protected from protease by the inner membrane. Purified mitochondria or mitoplasts containing Mss2p-HA were subjected to digestion with proteinase K. Samples were analyzed by using Western blotting with anti-HA, anti-cytochrome b₂, and anti-Arg8p.

FIG. 8

Mss2p has at least one TPR-like motif. Analysis of the Mss2p sequence by using the ProDom protein domain database (http://www.toulouse.inra.fr/prodom.html) indicated the presence of a TPR-like motif (12). This region of Mss2p is aligned with TPR motifs of Tom70p, Sti1p, and Pet117 (see Discussion). Boxed letters represent amino acid identity with Mss2p sequence. Shaded letters represent amino acid similarity with the Mss2p sequence. The gray bar above the sequences delimits the TPR-like motif in Mss2p.