cerevisiaestrains used in this study are listed inTable 1. processing byImp1occurs in the absence ofCox20andi-AAA protease activity, but is usually greatly reduced in efficiency. Under these conditions some matureCox2is usually put together into cytochromecoxidase allowing weak respiratory growth. Thus, theCox20chaperone has important functions in leader peptide processing, C-tail export, and stabilization ofCox2. CYTOCHROMEcoxidase is composed of three subunits encoded in the mitochondrial genome (mtDNA) and eight subunits encoded in the nuclear genome in the budding yeastSaccharomyces cerevisiae. In addition to the genes for these subunits, at least 20 other nuclear yeast genes are specifically required for synthesis of the mitochondrially TH588 coded subunits and post-translational actions in assembly of the active enzyme (Barrientoset al.2002;Herrmann and Funes 2005;Fontanesiet al.2006). The second largest subunit of cytochromecoxidase,Cox2, is usually a mitochondrial gene product whose acidic N-terminal and C-terminal domains are translocated through the inner membrane GTF2H from your matrix to the intermembrane space (IMS) and flank two transmembrane helices (Tsukiharaet al.1996).Cox2topogenesis is of particular interest since its hydrophilic domains are the largest known to be exported through the inner membrane. In budding yeast, localized membrane-bound translation of theCOX2mRNA, specifically activated byPet111(Green-Willmset al.2001;Naithaniet al.2003), produces a precursor, pre-Cox2, with a short N-terminal leader peptide (Pratjeet al.1983). The pre-Cox2N-tail is usually co-translationally exported byOxa1(He and Fox 1997;Hellet al.1998), a highly conserved inner membrane translocase that is also required for C-tail export (reviewed inBonnefoyet al.2009). Once in the IMS, the pre-Cox2leader peptide is rapidly processed by TH588 the inner membrane protease (IMP) (Nunnariet al.1993;Janet al.2000), in a reaction chaperoned byCox20(Hellet al.2000). The acidicCox2C-tail is usually exported to the IMS by a mechanism that is unique from N-tail export and appears to be post-translational (He and Fox 1997;Fiumeraet al.2007). C-tail export depends specifically upon another highly conserved inner membrane translocase,Cox18(Saracco and Fox 2002), which is usually paralogously related toOxa1, as well as to bacterial YidC proteins (Funeset al.2004). In addition,Cox2C-tail export requiresMss2and is usually promoted byPnt1, two proteins that interact withCox18(He and Fox 1999;Broadleyet al.2001;Saracco and Fox 2002). Interestingly, overproduction ofOxa1in a mutant lackingCox18results in some export of theCox2C-tail, althoughCox2remains unassembled and the cells fail to respire (Fiumeraet al.2009). This result suggested thatCox18has an assembly function in the IMS that overproducedOxa1cannot carry out, in addition to its translocation function. One possibility here is thatCox18could promote conversation of the exportedCox2C-tail with an assembly factor in the IMS. One candidate for such a factor isCox20. Cox20has previously been shown to be a 205-amino-acid integral mitochondrial inner TH588 membrane protein. It has two centrally located transmembrane helices flanked by hydrophilic domains in the intermembrane space (Hellet al.2000).Cox20interacts directly with pre-Cox2and promotes its processing. Since this conversation depends upon export of pre-Cox2byOxa1, it appears to involve domains in the IMS (Hellet al.2000). In addition,Cox20remains associated with unassembled matureCox2, suggesting that it has functions in cytochromecoxidase assembly downstream of pre-Cox2processing (Hellet al.2000;Preusset al.2001;Herrmann and Funes 2005), possibly includingCox2metallation (Rigbyet al.2008).Cox20has therefore been described as a chaperone, although it has no detectable similarity to other well-characterized chaperones or domains of known function. TH588 In this study we have investigated the role ofCox20in the export and assembly ofCox2. We find thatCox20is required for efficient export of theCox2C-tail and that it interacts with the translocaseCox18but only whenCox2is usually present. In addition,Cox20stabilizes mature but unassembledCox2. == Materials and Methods == == Yeast strains and genetic analysis of pseudorevertants == S. cerevisiaestrains used in this study are outlined inTable 1. All strains are congenic to D273-10B (ATCC 25657). Nuclear genes were manipulated using standard methods (Guthrie and Fink 1991). Transformation of plasmids and PCR products into yeast was accomplished with the EZ transformation kit (Zymo Research). Complete media (YPA) made up of adenine, dextrose (D), ethanol plus glycerol (EG), or raffinose (R) were prepared as previously explained (Guthrie and Fink 1991). Total synthetic media (CSM) and CSM lacking specific growth factors were purchased from Bio101 Systems.COX20was modified to encode a protein tagged with three MYC epitope at its C terminus, but with no other changes, by the pop-in pop-out strategy as explained (Schneideret al.1995). The plasmid pOXA1-W56R-ADH1(Supekovaet al.2010) was obtained from F. Supek and P. G. Schultz == Table 1 . Strains and plasmids used in this study. == Indie spontaneous pseudorevertants of thecox20 strain LEE83 were isolated by plating.
cerevisiaestrains used in this study are listed inTable 1
