INVESTIGATION Multiple Roles of the Cox20 Chaperone in Assembly of Saccharomyces cerevisiae Cytochrome c Oxidase Leah E. Elliott, Scott A. Saracco1, and Thomas D. Fox2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853 ABSTRACT The Cox2 subunit of Saccharomyces cerevisiae cytochrome c oxidase is synthesized in the mitochondrial matrix as a pre- cursor whose leader peptide is rapidly processed by the inner membrane protease following translocation to the intermembrane space. Processing is chaperoned by Cox20, an integral inner membrane protein whose hydrophilic domains are located in the intermembrane space, and Cox20 remains associated with mature, unassembled Cox2. The Cox2 C-tail domain is exported post-translationally by the highly conserved translocase Cox18 and associated proteins. We have found that Cox20 is required for efficient export of the Cox2 C-tail. Furthermore, Cox20 interacts by co-immune precipitation with Cox18, and this interaction requires the presence of Cox2.We therefore propose that Cox20 binding to Cox2 on the trans side of the inner membrane accelerates dissociation of newly exported Cox2 from the Cox18 translocase, promoting efficient cycling of the translocase. The requirement for Cox20 in cytochrome c oxidase assembly and respiratory growth is partially bypassed by yme1, mgr1 or mgr3 mutations, each of which reduce i-AAA protease activity in the intermembrane space. Thus, Cox20 also appears to stabilize unassembled Cox2 against degradation by the i-AAA protease. Pre- Cox2 leader peptide processing by Imp1 occurs in the absence of Cox20 and i-AAA protease activity, but is greatly reduced in efficiency. Under these conditions some mature Cox2 is assembled into cytochrome c oxidase allowing weak respiratory growth. Thus, the Cox20 chaperone has important roles in leader peptide processing, C-tail export, and stabilization of Cox2. YTOCHROME c oxidase is composed of three subunits bound translation of the COX2 mRNA, specifically activated Cencoded in the mitochondrial genome (mtDNA) and by Pet111 (Green-Willms et al. 2001; Naithani et al. 2003), eight subunits encoded in the nuclear genome in the bud- produces a precursor, pre-Cox2, with a short N-terminal ding yeast Saccharomyces cerevisiae. In addition to the genes leader peptide (Pratje et al. 1983). The pre-Cox2 N-tail is for these subunits, at least 20 other nuclear yeast genes are co-translationally exported by Oxa1 (He and Fox 1997; Hell specifically required for synthesis of the mitochondrially et al. 1998), a highly conserved inner membrane translocase coded subunits and post-translational steps in assembly of that is also required for C-tail export (reviewed in Bonnefoy the active enzyme (Barrientos et al. 2002; Herrmann and et al. 2009). Once in the IMS, the pre-Cox2 leader peptide is Funes 2005; Fontanesi et al. 2006). rapidly processed by the inner membrane protease (IMP) The second largest subunit of cytochrome c oxidase, Cox2, (Nunnari et al. 1993; Jan et al. 2000), in a reaction chaper- is a mitochondrial gene product whose acidic N-terminal and oned by Cox20 (Hell et al. 2000). C-terminal domains are translocated through the inner mem- The acidic Cox2 C-tail is exported to the IMS by a mech- brane from the matrix to the intermembrane space (IMS) and anism that is distinct from N-tail export and appears to be flank two transmembrane helices (Tsukihara et al. 1996). post-translational (He and Fox 1997; Fiumera et al. 2007). Cox2 topogenesis is of particular interest since its hydrophilic C-tail export depends specifically upon another highly con- domains are the largest known to be exported through the served inner membrane translocase, Cox18 (Saracco and inner membrane. In budding yeast, localized membrane- Fox 2002), which is paralogously related to Oxa1, as well Copyright © 2012 by the Genetics Society of America as to bacterial YidC proteins (Funes et al. 2004). In addition, doi: 10.1534/genetics.111.135665 Cox2 C-tail export requires Mss2 and is promoted by Pnt1, Manuscript received October 10, 2011; accepted for publication November 2, 2011 1Present address: Monsanto Company, 700 Chesterfield Pkwy, W, Chesterfield, MO two proteins that interact with Cox18 (He and Fox 1999; 63017. Broadley et al. 2001; Saracco and Fox 2002). Interestingly, 2Corresponding author: Department of Molecular Biology and Genetics, 333 Biotechnology Bldg., Cornell University, Ithaca, NY 14853-2703. E-mail: tdf1@cornell. overproduction of Oxa1 in a mutant lacking Cox18 results in edu some export of the Cox2 C-tail, although Cox2 remains Genetics, Vol. 190, 559–567 February 2012 559 unassembled and the cells fail to respire (Fiumera et al. determine that the mutations were recessive and to score 2009). This result suggested that Cox18 has an assembly their ability to complement the known genes. function in the IMS that overproduced Oxa1 cannot carry Analysis of mitochondrial proteins out, in addition to its translocation function. One possibility here is that Cox18 could promote interaction of the exported To examine export of the C terminus of Cox2, we employed Cox2 C-tail with an assembly factor in the IMS. One candi- strains whose mtDNA encodes a version of Cox2 bearing date for such a factor is Cox20. C-terminal HA-epitopes (Saracco and Fox 2002). Mitoplasts Cox20 has previously been shown to be a 205-amino-acid were prepared by osmotic shock from purified mitochondria integral mitochondrial inner membrane protein. It has two as described (Glick 1995; Glick and Pon 1995). For each centrally located transmembrane helices flanked by hydro- sample of mitoplasts, the equivalent of 75 mg of mitochondrial philic domains in the intermembrane space (Hell et al. proteinwastreatedwith20mg/ml proteinase K, or mock 2000). Cox20 interacts directly with pre-Cox2 and promotes treated, as described (Saracco and Fox 2002), except that its processing. Since this interaction depends upon export of after protease treatment samples were directly resuspended pre-Cox2 by Oxa1, it appears to involve domains in the IMS in 20 mM HEPES pH 7.4, 0.6 M sorbitol, 10% trichloroacetic (Hell et al. 2000). In addition, Cox20 remains associated acid, and 2 mM PMSF and incubated at 60° for 10 min. with unassembled mature Cox2, suggesting that it has roles For co-immune precipitation experiments, crude mito- in cytochrome c oxidase assembly downstream of pre-Cox2 chondria were prepared as described (Diekert et al. 2001). processing (Hell et al. 2000; Preuss et al. 2001; Herrmann One milligram of mitochondrial protein was solubilized in and Funes 2005), possibly including Cox2 metallation 1 ml of 1% digitonin, 100 mM NaCl, 20 mM Tris pH 7.4, and (Rigby et al. 2008). Cox20 has therefore been described as 1 mM PMSF for 10 min on ice and then centrifuged at a chaperone, although it has no detectable similarity to 16,000 · g for 10 min at 4°. Five percent of the clarified other well-characterized chaperones or domains of known supernatant was precipitated using Strataclean resin function. (StrataGene). The remaining supernatant was incubated In this study we have investigated the role of Cox20 in the with anti-hemagglutinin (anti-HA) antibody conjugated export and assembly of Cox2.Wefind that Cox20 is required beads (Roche). After incubation for 1 hr at 4°, the beads for efficient export of the Cox2 C-tail and that it interacts with were washed three times in the digitonin buffer before pro- the translocase Cox18 but only when Cox2 is present. In ad- teins were eluted in 5· Laemmli SDS sample buffer. dition, Cox20 stabilizes mature but unassembled Cox2. Protein samples to be analyzed by Western blotting were separated on 12% or 16% SDS-PAGE gels as indicated Materials and Methods and transferred to Immobilon-P polyvinylidene difluoride membrane (Millipore). Antibodies were anti-HA monoclonal Yeast strains and genetic analysis of pseudorevertants antibody clone 3F10 (Roche) (except the experiment of Fig- S. cerevisiae strains used in this study are listed in Table 1. ure 1, where anti-HA-HRP conjugate (Roche) was used), All strains are congenic to D273-10B (ATCC 25657). Nuclear anti-c-myc (anti-Myc) monoclonal antibody clone 9E10 genes were manipulated using standard methods (Guthrie (Roche), polyclonal anti- Cit1 (citrate synthase), polyclonal and Fink 1991). Transformation of plasmids and PCR prod- anti-Yme1, and monoclonal anti-Cox2 CCO6. Anti-mouse ucts into yeast was accomplished with the EZ transformation IgG-HRP (BioRad) or anti-rabbit IgG-HRP (BioRad) second- kit (Zymo Research). Complete media (YPA) containing ad- ary antibodies were applied after washing and detected by enine, dextrose (D), ethanol plus glycerol (EG), or raffinose the ECL and ECL+ detection methods (GE Healthcare). (R) were prepared as previously described (Guthrie and In vivo pulse-labeling of mitochondrial translation prod- Fink 1991). Complete synthetic media (CSM) and CSM lack- ucts was carried out as described (Bonnefoy et al. 2001) in ing specific growth factors were purchased from Bio101 Sys- cells grown on YPAR medium, except that there was no tems. COX20 was modified to encode a protein tagged with chase incubation, and mitochondria were prepared as de- three MYC epitope at its C terminus, but with no other scribed (Diekert et al. 2001). Radiolabeled mitochondrial changes, by the pop-in pop-out strategy as described proteins were separated on 16% SDS PAGE gels, which were (Schneider et al. 1995). The plasmid pOXA1-W56R-ADH1 dried and autoradiographed. (Supekova et al. 2010) was obtained from F. Supek and P. G. Schultz Results Independent spontaneous pseudorevertants of the cox20Δ strain LEE83 were isolated by plating cells from dis- Cox20 is required for Cox2 C-tail export independently of cytochrome c oxidase assembly or Cox2 tinct clonal cultures on YPAEG plates and incubating at 30° N-tail processing for 7 to 10 days. A single pseudorevertant was picked from each clonal culture, purified, and mated to cox20D (SCS194), Cox20 is known to be required for the N-terminal processing cox20Dmgr1D (LEE145), cox20Dmgr3D (LEE100), and of pre-Cox2 and to remain associated with mature Cox2 cox20Dyme1D (LEE106).