Báo cáo khoa học: Saccharomyces cerevisiae Cox18 complements the essential Sec-independent function of Escherichia coli YidC doc

Saccharomyces cerevisiae Cox18 complements the essential Sec-independent function of Escherichia coli YidC Edwin van Bloois 1 , Gregory Koningstein 1 , Heike Bauerschmitt 2,3 , Johannes M. Herrmann 2 and Joen Luirink 1 1 Department of Molecular Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, Amsterdam, the Netherlands 2 Zellbiologie, Technische Universita ¨ t Kaiserslautern, Germany 3 Institut fu ¨ r Physiologische Chemie, Universita ¨ tMu ¨ nchen, Germany Conserved mechanisms are used for the targeting and assembly of membrane proteins in bacteria, mitochon- dria and chloroplasts. This is illustrated by the con- served YidC ⁄ Oxa1 ⁄ Alb3 protein family that constitutes a class of proteins involved in the biogenesis of proteins of the bacterial inner membrane, mitochondrial inner membrane and chloroplast thylakoid membrane. YidC functions in the biogenesis of inner membrane proteins (IMPs) in Escherichia coli, both in concert with the Sec-translocon and as a Sec-independent insertase (reviewed in [1,2]). The Sec-translocon works as a protein-conducting channel for secretory proteins and IMPs. The translocon may include multiple compo- nents, but the core is formed by a heterotrimeric complex of SecY, SecE and SecG [3]. The translocation of secretory proteins and larger periplasmic domains of IMPs is driven by the ATPase SecA, which is peripher- ally associated with the SecYEG complex [4]. In con- trast, many IMPs do not rely on the activity of SecA, but are inserted in a cotranslational process which requires the signal recognition particle (SRP) and its receptor FtsY. The SRP binds to hydrophobic target- ing signals present in nascent IMPs, typically trans- membrane segments (TMs), and the ribosome nascent chain-SRP complex is then transferred to the Sec-trans- locon via membrane-associated FtsY [5]. The role of YidC in the context of the translocon is currently not well understood and may be versatile. Keywords Cox18; Oxa1; SecYEG; signal recognition particle; YidC Correspondence J. Luirink, Department of Molecular Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands Fax: + 31-20-5987155 Tel: + 31-20-5987175 E-mail: joen.luirink@falw.vu.nl (Received 27 July 2007, revised 31 August 2007, accepted 4 September 2007) doi:10.1111/j.1742-4658.2007.06094.x Members of the YidC ⁄ Oxa1 ⁄ Alb3 protein family function in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. In Esc- herichia coli, YidC plays a key role in the integration and assembly of many inner membrane proteins. Interestingly, YidC functions both in con- cert with the Sec-translocon and as a separate insertase independent of the translocon. Mitochondria of higher eukaryotes contain two distant homo- logues of YidC: Oxa1 and Cox18 ⁄ Oxa2. Oxa1 is required for the insertion of membrane proteins into the mitochondrial inner membrane. Cox18 ⁄ Oxa2 plays a poorly defined role in the biogenesis of the cytochrome c oxidase complex. Employing a genetic complementation approach by expressing the conserved region of yeast Cox18 in E. coli, we show here that Cox18 is able to complement the essential Sec-independent function of YidC. This identifies Cox18 as a bona fide member of the YidC ⁄ Oxa1 ⁄ Alb3 family. Abbreviations EcCox18, E. coli-targeted Cox18; HA, haemagglutinin; IMP, inner membrane protein; IMV, inner membrane vesicle; IPTG, isopropyl thio-b- D- galactoside; Lep, leader peptidase; LHCP, light-harvesting chlorophyll a ⁄ b binding protein; MscL, mechanosensitive channel of large conductance; PMF, proton motive force; PspA, phage shock protein A; RBD, ribosome binding domain; SRP, signal recognition particle; TM, transmembrane segment. 5704 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS Pull-down experiments have identified YidC as a Sec- associated component [6–8]. In vitro cross-link studies suggest that YidC operates downstream of the Sec- translocon for several complex IMPs to assist the transfer and assembly of TMs into the lipid bilayer [9– 12]. Remarkably, YidC seems to be largely dispensable for the insertion of Sec-dependent IMPs per se, imply- ing a later role of YidC in the biogenesis of IMPs [9,13–15]. Consistently, YidC has recently been shown to function in folding of the polytopic IMP LacY [16]. By contrast, YidC is absolutely required for the inser- tion of most Sec-independent IMPs. Substrates of this ‘YidC-only’ pathway include the phage coat proteins M13 and Pf3 and the endogenous IMPs F o c (subunit of the F 1 F 0 -ATPase complex) and MscL (mechanosen- sitive channel of large conductance) [17–22]. Consistent with an additional, Sec-independent function, YidC is present in excess when compared with Sec-translocon components [23]. The mitochondria of plants, fungi and animals harbour two distant YidC homologues: Oxa1 and Cox18 ⁄ Oxa2 [24]. Both Oxa1 and Cox18 ⁄ Oxa2 func- tion in the assembly of respiratory chain complexes, but are apparently nonredundant, as mutants in one component cannot be complemented by overexpression of the other [24]. The Oxa1 protein of Saccharomyces cerevisiae is the founding member of the YidC ⁄ Oxa1 ⁄ Alb3 family, and was originally identified as a factor involved in the biogenesis of respiratory chain complexes [25,26]. Oxa1 is required for the insertion of mitochondrially encoded proteins into the inner mem- brane, in particular subunits of the cytochrome c oxi- dase and ATPase complex, as well as for the insertion of some nuclear encoded proteins [26–31]. Cox18 was originally identified as a component required for bio- genesis or stability of cytochrome c oxidase [32,33]. In an independent genetic analysis, Cox18 ⁄ Oxa2 was found to be required for the efficient topogenesis of Cox2, one of the central subunits of the cytochrome c oxidase complex [34]. Cox18 ⁄ Oxa2 thereby directly interacts with Cox2 [24], but its molecular role is not clear. Although predictions suggest significant similar- ity in secondary structure and topology between YidC, Oxa1 and Cox18 ⁄ Oxa2, the overall level of primary sequence conservation between these proteins is low (15–20%). A conserved function is therefore not imme- diately obvious, especially as Cox18-deficient strains cannot be complemented by Oxa1 overexpression. We have reported previously that, despite the low degree of sequence similarity, Oxa1 can complement a YidC defect in E. coli to some extent, and vice versa, sug- gesting functional conservation between Oxa1 and YidC [35,36]. In this work, we address the functional correlation between yeast Cox18 and YidC using a similar genetic complementation approach in E. coli. The data demonstrate that Cox18 complements the essential Sec-independent function of YidC. Hence, by this criterion, Cox18 can be considered a bona fide member of the YidC ⁄ Oxa1 ⁄ Alb3 family. Results Properties of YidC, Oxa1 and Cox18 Members of the YidC ⁄ Oxa1 ⁄ Alb3 family are charac- terized by a conserved hydrophobic core domain of approximately 200 residues comprising five predicted TMs (Fig. 1A) [1]. The core domains of bacterial and mitochondrial homologues are flanked by nonrelated regions. The E. coli core domain is preceded by a large periplasmic loop and an additional TM. Mitochondrial Oxa1 and Cox18 lack this region, but are initially synthesized with an N-terminal presequence that is required for mitochondrial import and is cleaved from the mature protein in the matrix [24,29]. Downstream of the last TM, Oxa1 has an extended C-terminal domain of approximately 90 residues that protrudes into the matrix and functions as a ribosome binding domain (RBD) [37,38]. This domain is not conserved in YidC and Cox18. To study the functioning of Cox18 in E. coli, a hybrid protein was constructed consisting of the first 247 amino acids of YidC fused to the mature part of yeast Cox18 (residues 35–316), and was named Ec- Cox18 for E. coli-targeted Cox18 (Fig. 1B). The YidC portion was fused to mature Cox18 to enable mem- brane targeting of the hybrid, and contains: (a) TM1, which functions as an uncleaved signal sequence [23], and (b) part of the first periplasmic loop. Importantly, the periplasmic loop is not conserved, and over 90% can be deleted without a loss of function. The C-termi- nal region of the periplasmic loop is crucial for YidC activity (residues 323–346) [8], but this portion is not included in our fusion construct. Previously, we stud- ied the functioning of Oxa1 in E. coli using a similar fusion construct, termed EcOxa1 [35], that is included in the present work as a positive control. Both fusion constructs were cloned into the medium- and low-copy isopropyl isopropyl thio-b-d-galactoside (IPTG)-induc- ible expression plasmids pEH1 and pCL1921. EcCox18 complements the growth defect of a YidC depletion strain To examine whether EcCox18 is able to complement growth in the absence of YidC, EcCox18 was E. van Bloois et al. Evolutionarily conserved function of S. cerevisiae Cox18 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS 5705 expressed from a plasmid in strain JS7131 in which the yidC gene is under arabinose promoter control. The empty vector and an EcOxa1 expression plasmid were used as controls. EcOxa1 has previously been shown to complement the growth of JS7131 on depletion of YidC [35]. Cells were grown until mid-log phase, when tenfold serial dilutions were prepared and spotted onto solid medium as indicated (Fig. 2). Cells grown in the presence of arabinose to induce endogenous yidC expression showed normal growth as expected (Fig. 2A). In the absence of arabinose, no growth was observed unless expression of EcCox18 (or EcOxa1) was induced by the presence of IPTG in the growth medium (Figs 2B,C). Apparently, like EcOxa1, expres- sion of EcCox18 can efficiently restore the growth of JS7131 depleted for YidC, arguing that Cox18 can functionally complement the depletion of YidC under these conditions. To verify the expression of EcOxa1 and EcCox18, the strains used in Fig. 2 were grown in liquid medium and analysed by western blotting employing antiserum against YidC. In the presence of arabinose, the expres- sion of YidC is observed in cells harbouring the empty vector, whereas no YidC is detected in the absence of arabinose (Fig. 3A, lanes 1 and 2). In the presence of A B C Fig. 2. EcCox18 complements the growth of the YidC depletion strain JS7131. JS7131 containing EcCox18 in the expression plas- mid pEH1, or the empty vector or EcOxa1 in the expression plas- mid pEH1 (as controls), was grown to mid-log phase in liquid LB medium. Tenfold serial dilutions of the cultures were prepared and spotted onto LB plates supplemented with 0.2% L-arabinose (A) or 50 l M IPTG (C) to induce expression of either the chromosomal yidC or the plasmid-encoded fusion constructs. To deplete cells for YidC and minimize expression of the fusion constructs, arabinose and IPTG were omitted from the plates (B). The plates were incu- bated overnight at 37 °C. A B Fig. 1. Characteristics of E. coli YidC and its mitochondrial homo- logues Oxa1 and Cox18. (A) Membrane topology of YidC and mature Oxa1 and Cox18. The Oxa1 and Cox18 proteins are initially synthesized with a matrix targeting sequence which is proteolyti- cally removed on import. Mature Oxa1 and Cox18 span the mito- chondrial inner membrane five times. Oxa1 has an extended ribosome binding domain (RBD) at its C-terminus that protrudes into the matrix. E. coli YidC has six transmembrane segment (TMs), the first of which functions as an uncleaved signal sequence. (B) Schematic representation of the Cox18 (EcCox18) and Oxa1 (EcOxa1) fusion constructs used in this study. The con- structs comprise mature Cox18 (residues 35–316), or Oxa1 (resi- dues 43–402), fused to the N-terminal targeting domain of YidC (residues 1–247). A linker sequence (hatched area) is included in the Oxa1 hybrid. Evolutionarily conserved function of S. cerevisiae Cox18 E. van Bloois et al. 5706 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS IPTG, but absence of arabinose, the fusion constructs EcOxa1 and EcCox18 are exclusively detected (Fig. 3A, lanes 3 and 4), confirming that, under these conditions, EcOxa1 and EcCox18, but not YidC, are expressed. Furthermore, using ultracentrifugation to separate cell fractions (data not shown), we confirmed that, like YidC, ExCox18 is localized in the inner membrane. The depletion of YidC has been shown to rapidly induce the expression of phage shock protein A (PspA), a stress protein that responds to a dissipation of the proton motive force (PMF). This, in turn, is caused by defects in the membrane assembly of the cytochrome o oxidase and F 1 F 0 -ATPase complexes, which appear to be a direct consequence of YidC depletion [39]. To monitor whether EcCox18 is able to suppress the PspA response in the absence of YidC, PspA levels of the cell samples described above were analysed by western blotting (Fig. 3B). In the absence of YidC, increased amounts of PspA are detected in cells harbouring the empty vector when compared with the same cells not depleted for YidC (Fig. 3B, lanes 1 and 2). Consistent with previous data, the PspA response is suppressed by expression of EcOxa1 (Fig. 3B, lane 3) [35]. Similarly, no PspA is detected in cells depleted for YidC but expressing EcCox18 (Fig. 3B, lane 4). This suggests that EcCox18 supports the correct membrane assembly of E. coli respiratory chain complexes, thus sustaining PMF. EcCox18 functions as a Sec-independent insertase Some IMPs or domains of IMPs do not require the Sec-translocon for insertion, but are directly inserted via YidC [17–22,40–42]. To test whether EcCox18 can replace YidC in this Sec-independent pathway, we analysed its ability to mediate membrane insertion of M13P2. M13P2 is an M13 procoat derivative that is extended at its C-terminus with the P2 domain of lea- der peptidase (Lep; Fig. 4A). M13P2 is synthesized with a signal sequence that is processed by signal peptidase I. Processing and membrane integration of M13P2 is, like wild-type M13 procoat, strictly depen- dent on YidC, but not on the Sec-machinery [14,18,43]. Here, M13P2 and EcCox18 were expressed in the conditional yidC strain JS7131 from compatible plasmids. Processing of M13P2 was monitored on pulse labelling in order to clearly distinguish between the precursor and mature forms. In the presence of YidC, precursor M13P2 is converted into its mature form (Fig. 4B, upper panel, lane 1). In the absence of YidC, the processing of the precursor is strongly affected (Fig. 4B, upper panel, lane 2), as observed previously. However, efficient processing is completely restored when EcCox18 is expressed instead of YidC (Fig. 4B, upper panel, lane 3), suggesting that EcCox18 can mediate the insertion and translocation of M13P2. To verify the depletion of YidC and expres- sion of EcCox18 under these conditions, cell samples taken prior to pulse labelling were analysed by western blotting (Fig. 4B, bottom panel). To further confirm Sec-independent functioning of EcCox18, we analysed its ability to insert the N-terminal domain of pre-CyoA. CyoA is a subunit of the cyto- chrome o oxidase that is synthesized with a lipoprotein- type signal sequence (Fig. 4C). YidC is sufficient to mediate translocation of the N-terminal periplasmic loop and processing of the signal peptide by the lipopro- tein-specific signal peptidase II [40–42]. These initial steps are also a prerequisite for translocation of the large C-terminal domain that involves both the Sec-translo- con and SecA. Here, we analysed the processing of hae- magglutinin (HA)-tagged pre-CyoA under steady state conditions to indicate insertion and translocation of the N-terminal domain (Fig. 4D). Pre-CyoA and EcCox18 were expressed in the conditional yidC strain JS7131 from compatible plasmids. As shown previously, pro- cessing of pre-CyoA is severely affected on depletion of YidC (Fig. 4D, upper panel, lanes 1 and 2). Efficient processing is completely restored when EcCox18 is expressed instead of YidC (Fig. 4D, upper panel, lane 3), suggesting that EcCox18 can mediate the A B Fig. 3. EcCox18 is expressed and suppresses the PspA response in the YidC depletion strain JS7131. JS7131 cells harbouring the constructs indicated in Fig. 2 were grown to mid-log phase in liquid LB medium in the presence of 0.2% L-arabinose (lane 1) or 50 lM IPTG (lanes 3 and 4) to induce expression of either the chromo- somal yidC or the plasmid-encoded fusion constructs. Arabinose and IPTG were omitted from the culture medium to deplete cells for YidC (lane 2). Cell samples were taken, and 0.1 D 660 units of cells were analysed by SDS-PAGE and western blotting, using anti- serum against YidC (A) or PspA (B). E. van Bloois et al. Evolutionarily conserved function of S. cerevisiae Cox18 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS 5707 insertion and translocation of the N-terminus of pre- CyoA. Remarkably, the processing of pre-CyoA is most efficient in the presence of EcCox18. Most probably, this relates to a slightly higher expression of plasmid- encoded EcCox18 compared with endogenous YidC expression (Fig. 4D, bottom panel). Together, these data demonstrate that EcCox18 mediates the efficient insertion of YidC-dependent ⁄ Sec- independent IMPs, consistent with its ability to restore growth in the absence of YidC expression. EcCox18 is not cross-linked to nascent chains of Sec-dependent IMPs Although YidC appears to be dispensable for the membrane insertion of Sec-dependent IMPs, it has been shown to function at a later stage in the biogene- sis of IMPs, assisting in the folding and assembly into their native structure [16]. This role is reflected in rela- tively late contacts between YidC and nascent Sec- dependent IMPs, as evidenced by photo-cross-linking studies [6,9,10,12]. To examine whether Cox18 is able to participate in this Sec-dependent pathway, we analy- sed the putative contacts between EcCox18 and sub- strates of this pathway that are trapped during integration in the inner membrane. Initially, we analysed interactions of the Sec-depen- dent IMP FtsQ by photo-cross-linking. Radiolabelled nascent chains of FtsQ with a length of 108 residues were generated by in vitro translation of truncated mRNA in a cell-free translation system in the presence of [ 35 S]methionine. During translation, a photoreactive cross-linking probe was specifically incorporated into the nascent chains at position 40 in the TM (Fig. 5A). Inner membrane vesicles (IMVs) derived from JS7131 cells that express EcCox18 or YidC as a control (see Fig. 5C for YidC ⁄ EcCox18 content; a presumed Ec- Cox18 degradation product is indicated by an arrow- head) were added to allow cotranslational targeting of the translation intermediate. Subsequently, the cross-linking probe was activated by UV irradiation, followed by sodium carbonate extraction to recover membrane-integrated material. Almost exclusive cross- linking to YidC and SecY was observed when control A B C D Fig. 4. EcCox18 promotes membrane insertion of M13P2 and CyoA. (A) Membrane topology model of M13P2. M13P2 is an M13 procoat derivative that is extended at its C-terminus with the P2 domain of Lep. M13P2 is synthesized with a signal sequence that is processed by signal peptidase I (SPaseI) (arrow). (B) Pulse-label analysis of M13P2 processing in strain JS7131 harbouring an Ec- Cox18 expression plasmid or, as control, the empty vector. Cells were grown and processed as described in Experimental proce- dures. Prior to pulse labelling, a cell sample was taken and analy- sed by SDS-PAGE and western blotting using antiserum against YidC (lower panel). The precursor and mature forms are denoted as ‘p’ and ‘m’, respectively. The signal sequence is represented by a solid white bar and TMs are represented by solid black bars. (C) Membrane topology model of CyoA-HA. CyoA is synthesized with a lipoprotein-type signal sequence that is processed by SPaseII (arrow). Mature CyoA comprises two TMs connected by a small cytoplasmic loop and two translocated termini: a lipid-modified N-terminus and a large C-terminus. To permit immunodetection, an HA tag was attached to the C-terminus. (D) Steady state analysis of CyoA processing in strain JS7131 harbouring an EcCox18 expression plasmid or, as control, the empty vector. Cells were grown and processed as described in Experimental procedures. CyoA processing was analysed in 0.1 D 660 units of cells by SDS- PAGE and western blotting using HA antiserum. YidC ⁄ EcCox18 levels in the cells used (monitored as described in B) are shown in the lower panel. Evolutionarily conserved function of S. cerevisiae Cox18 E. van Bloois et al. 5708 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS IMVs were used (Fig. 5B, lane 1; see also [6]). The identity of the cross-linked adducts was confirmed by immunoprecipitation (Fig. 5B, lanes 7 and 8). Using IMVs that contain EcCox18 instead of YidC, no cross-linking product was observed in the  70 kDa region, whereas cross-linking to SecY was unaffected (Fig. 5B, lanes 4, 9 and 10). These data indicate that EcCox18 is not in the vicinity of the FtsQ signal anchor sequence during membrane insertion. The observed cross-linking to SecY suggests normal mem- brane insertion of FtsQ nascent chains under these conditions, confirming that YidC is not required for the initial insertion of FtsQ per se [15,35]. The lack of EcCox18 cross-linking indicates that EcCox18 is not located near the Sec-translocon. However, it cannot be excluded that the cross-linking probe in FtsQ is close to EcCox18, but rigidly oriented in another direction. To address this point further, we analysed the interac- tions of a second Sec-dependent nascent IMP, Lep with a length of 50 residues, and a cross-linking probe at position 10 or 15 in the first TM (Fig. 5A). As observed previously, 50LepTAG10 shows strong A C B Fig. 5. EcCox18 is not in close proximity to membrane-inserted nascent FtsQ and Lep during membrane insertion. (A) Schematic representa- tion of the FtsQ 108mer and Lep 50mer with cross-linking probes at position 40 (108FtsQ) and 10 or 15 (50Lep). TMs are represented by thick lines. (B) In vitro translation of 108FtsQTAG40, 50LepTAG10 and 50LepTAG15 was carried out in the presence of (Tmd)Phe-tRNA sup and IMVs derived from JS7131 cells expressing either YidC or EcCox18 as indicated. After translation, samples were irradiated with UV light to induce cross-linking and extracted with sodium carbonate to recover membrane-integrated material. The pellet fractions were either directly analysed (lanes 1–6) or immunoprecipitated (IP) using antiserum against YidC (lanes 7 and 9) or SecY (lanes 8 and 10). (C) The YidC ⁄ EcCox18 content of the IMVs used was analysed by SDS-PAGE and western blotting, using antiserum against YidC and Lep (control protein for IM localization). The position of EcCox18 is indicated by an asterisk. A presumed EcCox18 degradation product is indicated by an arrowhead. E. van Bloois et al. Evolutionarily conserved function of S. cerevisiae Cox18 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS 5709 cross-linking to YidC, whereas 50LepTAG15 predomi- nantly contacts SecY when control IMVs are used (Fig. 5B, lanes 2 and 3; see also [13]). No cross-linking to EcCox18 was observed using either construct, sug- gesting that the first TM of Lep is not in proximity to EcCox18 during membrane insertion (Fig. 5B, lanes 5 and 6). Together, these data suggest that EcCox18 does not contact nascent Sec-dependent IMPs, probably because of its inability to associate with the Sec-translocon that is notably absent in mitochondria [44]. Discussion Members of the YidC ⁄ Oxa1 ⁄ Alb3 protein family are involved in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. Complemen- tation studies have shown that, despite little similarity at the primary sequence level, the members of this family are, to a large extent, functionally conserved [35,36,45]. Despite this general exchangeability, mito- chondria of higher eukaryotes consistently contain a second Oxa1-like protein, Oxa2 ⁄ Cox18, that is unable to complement the function of Oxa1. Rather, it fulfils a specific function, presumably in the translocation of the C-terminal domain of Cox2 [24,27,34,46]. Here, we show that yeast Cox18, like Oxa1, can take over the essential Sec-independent function of YidC [35]. Vice versa, we have recently demonstrated that YidC can partly complement Cox18 functioning in mitochondria [36]. Together, these studies define Cox18 as a bona fide member of the YidC⁄ Oxa1 ⁄ Alb3 protein family, sharing an intrinsic insertase ⁄ translocase activity. The conclusion that Cox18 can take over the Sec- independent function of YidC is based on several observations. Firstly, expression of EcCox18 (Cox18 fused to the nonessential first TM and part of the first periplasmic loop of YidC) is able to sustain growth in the absence of YidC. Previous studies have shown that the Sec-independent function of YidC is essential for growth [35]. Secondly, EcCox18 is able to promote membrane insertion of the model IMP M13P2 and the N-terminus of the cytochrome o oxidase subunit CyoA, which have both been shown to insert via a YidC-dependent but Sec-independent mechanism [14,18,40–42]. Thirdly, EcCox18 expression completely suppresses the up-regulation of the stress protein PspA on depletion of YidC. PspA has been shown to respond to a dissipation of the PMF caused by defects in the assembly of respiratory chain complexes when YidC is depleted [39]. Notably, insertion of the F o c subunit of the F 1 F 0 -ATPase and of the N-terminus of CyoA (see above) is exclusively dependent on YidC. Consequently, F 1 F 0 -ATPase and cytochrome o oxidase activity are severely diminished when YidC is depleted. Apparently, both Oxa1 and Cox18 can functionally replace YidC in the ‘YidC-only’ pathway, suggesting that, despite distinct functions and interactions in mito- chondria, both components share a conserved inser- tase-like core activity. It is not clear how members of the YidC ⁄ Oxa1 ⁄ Alb3 family recognize their substrate proteins. In E. coli, the SRP has been implicated in tar- geting of the endogenous substrates F o c and MscL to YidC [18,22]. Moreover, in chloroplasts, a cpSRP ⁄ Alb3 pathway is operational and is used by light-harvesting chlorophyll a ⁄ b binding protein (LHCP). Consistently, a functional interaction between cpSRP, cpFtsY and Alb3 has been demonstrated [47,48]. Mitochondria have lost the SRP system and, in this case, cotransla- tional insertion is mediated by a physical contact of Oxa1 to ribosomes. However, Cox18 lacks the RBD and fails to bind ribosomes in mitochondria (E. van Bloois, H. Bauerschmitt & J. M. Herrmann, unpub- lished observations). Therefore, it is exciting to see that EcCox18 complements the YidC depletion strain, which suggests that, at least in the bacterial context, Cox18 can facilitate SRP-mediated protein insertion. The suggestion that Cox18 is unable to complement the Sec-dependent function of YidC is based on in vitro site-specific photo-cross-linking of nascent Sec- dependent IMPs synthesized in the presence of IMVs that contain EcCox18 instead of YidC. Clearly, Ec- Cox18 was not cross-linked to the nascent substrates from positions in the TM that were strongly cross- linked to YidC in wild-type IMVs. Strikingly, nascent FtsQ and Lep were still able to insert in these IMVs at SecY as in wild-type IMVs. These results are reminis- cent of the inability of EcOxa1 to replace YidC in this assay [35]. In addition, EcOxa1 appeared less able to complement the supporting role of YidC in folding of the Sec-dependent IMP LacY [35]. Most probably, both EcCox18 and EcOxa1 are unable to associate with the Sec-translocon and receive TM segments of nascent IMPs that insert at the Sec-translocon. Cox18 and Oxa1 have probably lost affinity for the Sec-trans- locon that is absent in mitochondria. Furthermore, the N-terminus of YidC that is fused in the EcCox18 and EcOxa1 constructs does not include the C-terminal region of the first periplasmic loop that has been impli- cated in connecting YidC to the Sec-translocon [8]. The biogenesis of both mitochondrial Cox2 and its bacterial homologue CyoA are complex, multistep pro- cesses for reasons that are not immediately obvious. Insertion of Cox2 requires the sequential action of two YidC family members, Oxa1 and Cox18, to ensure cor- rect translocation of the N- and C-terminus of Cox2 Evolutionarily conserved function of S. cerevisiae Cox18 E. van Bloois et al. 5710 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS [27,28,34,46]. Insertion of CyoA involves a sequential action of YidC to translocate the N-terminus and allow signal peptide processing and of the Sec-translocon to translocate the large C-terminal domain [40–42]. Possibly, Cox18 has specifically evolved to translocate the Cox2 C-terminus in mitochondria that have lost the Sec-translocon. Remarkably, Cox18 achieves this by using a conserved, YidC-type insertase activity. Experimental procedures Reagents, enzymes and sera The restriction enzymes, Expand long template PCR system and Lumi-Light PLUS western blotting substrate were obtained from Roche Molecular Biochemicals (Mannheim, Germany). [ 35 S]methionine and Protein A sepharose were purchased from Amersham Biosciences (Uppsala, Sweden). T4 ligase and alkaline phosphatase were obtained from Invi- trogen (Carlsbad, CA, USA). Pansorbin was purchased from Merck (Darmstadt, Germany). Megashort script T7 tran- scription kit was obtained from Ambion Inc. (Austin, TX, USA). All other chemicals were supplied by Sigma–Aldrich (Steinheim, Germany). Antisera against YidC, PspA and Lep have been described previously or were from our own collec- tion [6,35]. Antiserum against influenza HA was from Sigma. Strains, plasmids and growth conditions E. coli strain Top10F¢ (Invitrogen) was used for the cloning and maintenance of plasmid constructs. Strain MRE600 was used to prepare translation lysate for suppression of UAG stop codons in the presence of (Tmd)Phe-tRNA sup [6]. YidC depletion strains JS7131 and FTL10 were grown as described previously [17,35]. YidC depletion strain JS7131 was used for the preparation of IMVs, essentially as described previously [6]. All strains were routinely grown in Luria–Bertani (LB) medium with appropriate antibiotics. Plasmids pEH1, pEH1-EcOxa1, pASKIBA3-M13P2 and pASKIBA3-CyoAHA have been described previously [35,42,49]. For in vitro photo-cross-linking experiments, the previously described pC4Meth108FtsQTAG40, pC4Meth50LepTAG10 and pC4Meth50LepTAG15 were used [6,13]. Plasmid pEH1-EcCox18His is based on pEH1- EcOxa1His [35] and harbours the EcCox18 fusion construct with an N-terminal hexahistidinyl tag under the control of a lac-promoter. For construction of this plasmid, the sequence encoding residues 35–316 of Cox18 was PCR amplified using genomic DNA of S. cerevisiae W303. The PCR product was KpnI ⁄ SmaI digested and used to replace the KpnI ⁄ SmaI Oxa1 fragment of pEH1-EcOxa1, yielding pEH1-EcCox18His. For complementation experiments, plasmid pEH1-EcOxa1 was used to replace the KpnI ⁄ SmaI fragment by the corresponding fragment of pEH1- EcCox18His, giving rise to pEH1-EcCox18, thereby remov- ing the hexahistidinyl tag of the Cox18 hybrid. The plasmid pEH1-EcOxa1, which encodes the EcOxa1 fusion construct lacking the N-terminal hexahistidinyl tag, was constructed by replacing the EcoRV ⁄ AgeI fragment of pEH-EcOxa1His [35] with the corresponding fragment of pEH1-YidCX [35]. A low-copy EcCox18 plasmid was constructed by replac- ing the KpnI ⁄ SmaI fragment of pCL-EcOxa1.K m and pCL-EcOxa1.S m [35] with the KpnI ⁄ SmaI fragment of pEH1-EcCox18His, yielding pCL-EcCox18.K m and pCL- EcCox18.S m . The nucleotide sequences of all constructs were verified by DNA sequencing. In vivo processing assays JS7131 harbouring pASKIBA3-CyoAHA and pCL1921K m , or pCL-EcCox18.K m , was grown as described previously [35]. Briefly, cells were grown in liquid LB medium supple- mented with 0.2% arabinose or 1 mm of IPTG to induce the expression of either chromosomal yidC or plasmid-encoded EcCox18. To deplete cells of YidC, arabinose and IPTG were omitted from the culture medium. The expression of CyoA was induced for 5 min by adding anhydrotetracycline (500 ngÆmL )1 ) to the cultures. Subsequently, a cell sample was taken, resuspended in sample buffer and subjected to SDS-PAGE and western blotting as indicated. JS7131 har- bouring pASKIBA3-M13P2 and pCL1921K m , or pCL-Ec- Cox18.K m , was grown as described above. M13P2 expression was induced for 4 min by the addition of anhydrotetracycline (500 ngÆmL )1 ) and the cells were labelled with [ 35 S]methio- nine (30 lCiÆmL )1 ) for 2 min. Radiolabelled proteins were acid precipitated and M13P2 was immunoprecipitated with anti-Lep serum. Samples were analysed by SDS-PAGE and proteins were visualized by phosphorimaging. In vitro transcription, translation, targeting and cross-linking Truncated mRNA was prepared as described previously [6] from HindIII linearized pC4Meth constructs. In vitro trans- lation and cross-linking of nascent FtsQ and Lep deriva- tives carrying the photo-activatable amino acid (Tmd)Phe were carried out as described previously [6]. Targeting to IMVs and carbonate extraction have been described previ- ously [6,9]. Carbonate-soluble and insoluble fractions were acid precipitated or immunoprecipitated using anti-SecY or anti-YidC sera. Samples were analysed by SDS-PAGE, and proteins were visualized by phosphorimaging as described previously [6]. Acknowledgements We thank Corinne M. ten Hagen-Jongman for techni- cal assistance and Wouter Jong, Dirk-Jan Scheffers and E. van Bloois et al. Evolutionarily conserved function of S. cerevisiae Cox18 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS 5711 Samuel Wagner for critical reading of the manuscript. EB is supported by the Council for Chemical Sciences of the Netherlands Society for Scientific Research. References 1 Kiefer D & Kuhn A (2007) YidC as an essential and multifunctional component in membrane protein assem- bly. Int Rev Cytol 259, 113–138. 2 Luirink J, von Heijne G, Houben E & de Gier JW (2005) Biogenesis of inner membrane proteins in Escher- ichia coli. Annu Rev Microbiol 59, 329–355. 3 Osborne AR, Rapoport TA & van den Berg B (2005) Protein translocation by the Sec61 ⁄ SecY channel. Annu Rev Cell Dev Biol 21, 529–550. 4 Vrontou E & Economou A (2004) Structure and func- tion of SecA, the preprotein translocase nanomotor. Biochim Biophys Acta 1694, 67–80. 5 Luirink J & Sinning I (2004) SRP-mediated protein tar- geting: structure and function revisited. Biochim Biophys Acta 1694, 17–35. 6 Scotti PA, Urbanus ML, Brunner J, de Gier JW, von Heijne G, van der Does C, Driessen AJ, Oudega B & Luirink J (2000) YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase. EMBO J 19, 542–549. 7 Nouwen N & Driessen AJ (2002) SecDFyajC forms a heterotetrameric complex with YidC. Mol Microbiol 44, 1397–1405. 8 Xie K, Kiefer D, Nagler G, Dalbey RE & Kuhn A (2006) Different regions of the nonconserved large peri- plasmic domain of Escherichia coli YidC are involved in the SecF interaction and membrane insertase activity. Biochemistry 45, 13401–13408. 9 Urbanus ML, Scotti PA, Fro ¨ derberg L, Sa ¨ a ¨ f A, de Gier JWL, Brunner J, Samuelson JC, Dalbey RE, Oudega B & Luirink J (2001) Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC. EMBO Rep 2, 524–529. 10 Houben EN, ten Hagen-Jongman CM, Brunner J, Oud- ega B & Luirink J (2004) The two membrane segments of leader peptidase partition one by one into the lipid bilayer via a Sec ⁄ YidC interface. EMBO Rep 5, 970– 975. 11 Houben EN, Zarivach R, Oudega B & Luirink J (2005) Early encounters of a nascent membrane protein: speci- ficity and timing of contacts inside and outside the ribo- some. J Cell Biol 170, 27–35. 12 Beck K, Eisner G, Trescher D, Dalbey RE, Brunner J & Muller M (2001) YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immedi- ate proximity to the SecYE translocon and lipids. EMBO Rep 2, 709–714. 13 Houben ENG, Urbanus ML, van der Laan M, ten Hagen-Jongman CM, Driessen AJM, Brunner J, Oudega B & Luirink J (2002) YidC and SecY mediate membrane insertion of a type I transmembrane domain. J Biol Chem 277, 35880–35886. 14 Fro ¨ derberg L, Houben E, Samuelson JC, Chen M, Park SK, Phillips GJ, Dalbey R, Luirink J & De Gier JW (2003) Versatility of inner membrane protein biogenesis in Escherichia coli. Mol Microbiol 47, 1015–1027. 15 van der Laan M, Nouwen N & Driessen AJ (2004) SecYEG proteoliposomes catalyze the Deltaphi-depen- dent membrane insertion of FtsQ. J Biol Chem 279 , 1659–1664. 16 Nagamori S, Smirnova IN & Kaback HR (2004) Role of YidC in folding of polytopic membrane proteins. J Cell Biol 165, 53–62. 17 Samuelson JC, Chen MY, Jiang FL, Moller I, Wied- mann M, Kuhn A, Phillips GJ & Dalbey RE (2000) YidC mediates membrane protein insertion in bacteria. Nature 406, 637–641. 18 van Bloois E, Jan Haan G, de Gier JW, Oudega B & Luirink J (2004) F(1)F (0) ATP synthase subunit c is targeted by the SRP to YidC in the E. coli inner mem- brane. FEBS Lett 576, 97–100. 19 Yi L, Celebi N, Chen M & Dalbey RE (2004) Sec ⁄ SRP requirements and energetics of membrane insertion of subunits a, b, and c of the Escherichia coli F1F0 ATP synthase. J Biol Chem 279, 39260–39267. 20 Serek J, Bauer-Manz G, Struhalla G, Van Den Berg L, Kiefer D, Dalbey R & Kuhn A (2004) Escherichia coli YidC is a membrane insertase for Sec-independent pro- teins. EMBO J 23, 294–301. 21 van der Laan M, Bechtluft P, Kol S, Nouwen N & Driessen AJ (2004) F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis. J Cell Biol 165, 213–222. 22 Facey SJ, Neugebauer SA, Krauss S & Kuhn A (2007) The mechanosensitive channel protein MscL is targeted by the SRP to the novel YidC membrane insertion pathway of Escherichia coli. J Mol Biol 365, 995–1004. 23 Urbanus ML, Fro ¨ derberg L, Drew D, Bjo ¨ rk P, de Gier JWL, Brunner J, Oudega B & Luirink J (2002) Target- ing, insertion, and localization of Escherichia coli YidC. J Biol Chem 277, 12718–12723. 24 Funes S, Nargang FE, Neupert W & Herrmann JM (2004) The Oxa2 protein of Neurospora crassa plays a critical role in the biogenesis of cytochrome oxidase and defines a ubiquitous subbranch of the Oxa1 ⁄ YidC ⁄ Alb3 protein family. Mol Biol Cell 15, 1853–1861. 25 Bauer M, Behrens M, Esser K, Michaelis G & Pratje E (1994) PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast. Mol Gen Genet 245, 272–278. 26 Bonnefoy N, Chalvet F, Hamel P, Slonimski PP & Dujardin G (1994) OXA1, a Saccharomyces cerevisiae nuclear gene whose sequence is conserved from Evolutionarily conserved function of S. cerevisiae Cox18 E. van Bloois et al. 5712 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS prokaryotes to eukaryotes controls cytochrome oxidase biogenesis. J Mol Biol 239, 201–212. 27 He S & Fox TD (1997) Membrane translocation of mi- tochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the con- served protein Oxa1p. Mol Biol Cell 8, 1449–1460. 28 Hell K, Herrmann J, Pratje E, Neupert W & Stuart RA (1997) Oxa1p mediates the export of the N- and C-ter- mini of pCoxII from the mitochondrial matrix to the intermembrane space. FEBS Lett 418, 367–370. 29 Herrmann JM, Neupert W & Stuart RA (1997) Inser- tion into the mitochondrial inner membrane of a poly- topic protein, the nuclear-encoded Oxa1p. EMBO J 16, 2217–2226. 30 Hell K, Neupert W & Stuart RA (2001) Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA. EMBO J 20, 1281– 1288. 31 Altamura N, Capitanio N, Bonnefoy N, Papa S & Dujardin G (1996) The Saccharomyces cerevisiae OXA1 gene is required for the correct assembly of cytochrome c oxidase and oligomycin-sensitive ATP synthase. FEBS Lett 382, 111–115. 32 Souza RL, Green-Willms NS, Fox TD, Tzagoloff A & Nobrega FG (2000) Cloning and characterization of COX18, a Saccharomyces cerevisiae PET gene required for the assembly of cytochrome oxidase. J Biol Chem 275, 14898–14902. 33 Hikkel I, Gbelska Y, van der Aart QJ, Lubecu G & Su- bik J (1997) Cloning and characterization of KlCOX18, a gene required for activity of cytochrome oxidase in Kluyveromyces lactis. Curr Genet 32, 267–272. 34 Saracco SA & Fox TD (2002) Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail and interacts with Pnt1p and Mss2p in the inner membrane. Mol Biol Cell 13, 1122– 1131. 35 van Bloois E, Nagamori S, Koningstein G, Ullers RS, Preuss M, Oudega B, Harms N, Kaback HR, Herrmann JM & Luirink J (2005) The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential. J Biol Chem 280, 12996–13003. 36 Preuss M, Ott M, Funes S, Luirink J & Herrmann JM (2005) Evolution of mitochondrial oxa proteins from bacterial YidC. Inherited and acquired functions of a conserved protein insertion machinery. J Biol Chem 280, 13004–13011. 37 Jia L, Dienhart M, Schramp M, McCauley M, Hell K & Stuart RA (2003) Yeast Oxa1 interacts with mito- chondrial ribosomes: the importance of the C-terminal region of Oxa1. EMBO J 22, 6438–6447. 38 Szyrach G, Ott M, Bonnefoy N, Neupert W & Herrmann JM (2003) Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria. EMBO J 22, 6448–6457. 39 van der Laan M, Urbanus ML, ten Hagen-Jongman CM, Nouwen N, Oudega B, Harms N, Driessen AJ & Luirink J (2003) A conserved function of YidC in the biogenesis of respiratory chain complexes. Proc Natl Acad Sci USA 100, 5801–5806. 40 du Plessis DJ, Nouwen N & Driessen AJ (2006) Subunit a of cytochrome o oxidase requires both YidC and Sec- YEG for membrane insertion. J Biol Chem 281, 12248– 12252. 41 Celebi N, Yi L, Facey SJ, Kuhn A & Dalbey RE (2006) Membrane biogenesis of subunit II of cytochrome b oxidase: contrasting requirements for insertion of N-ter- minal and C-terminal domains. J Mol Biol 357, 1428– 1436. 42 van Bloois E, Haan GJ, de Gier JW, Oudega B & Luir- ink J (2006) Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner mem- brane protein CyoA. J Biol Chem 281, 10002–10009. 43 Samuelson JC, Jiang F, Yi L, Chen M, de Gier JW, Kuhn A & Dalbey RE (2001) Function of YidC for the insertion of M13 procoat protein in E. coli: transloca- tion of mutants that show differences in their membrane potential dependence and Sec-requirement. J Biol Chem 276, 34847–34852. 44 Glick BS & von Heijne G (1996) Saccharomyces cerevi- siae mitochondria lack a bacterial-type Sec machinery. Protein Sci 5, 2651–2652. 45 Jiang F, Yi L, Moore M, Chen M, Rohl T, Van Wijk KJ, De Gier JW, Henry R & Dalbey RE (2002) Chloro- plast YidC homolog Albino3 can functionally comple- ment the bacterial YidC depletion strain and promote membrane insertion of both bacterial and chloroplast thylakoid proteins. J Biol Chem 277, 19281–19288. 46 Fiumera HL, Broadley SA & Fox TD (2007) Transloca- tion of mitochondrially synthesized Cox2 domains from the matrix to the intermembrane space. Mol Cell Biol 27, 4664–4673. 47 Moore M, Goforth RL, Mori H & Henry R (2003) Functional interaction of chloroplast SRP ⁄ FtsY with the ALB3 translocase in thylakoids: substrate not required. J Cell Biol 162, 1245–1254. 48 Moore M, Harrison MS, Peterson EC & Henry R (2000) Chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyll-binding protein into thylakoid membranes. J Biol Chem 275, 1529–1532. 49 Hashemzadeh-Bonehi L, Mehraein-Ghomi F, Mitsopou- los C, Jacob JP, Hennessey ES & Broome-Smith JK (1998) Importance of using lac rather than ara promoter vectors for modulating the levels of toxic gene products in Escherichia coli. Mol Microbiol 30, 676–678. E. van Bloois et al. Evolutionarily conserved function of S. cerevisiae Cox18 FEBS Journal 274 (2007) 5704–5713 ª 2007 The Authors Journal compilation ª 2007 FEBS 5713 . by expressing the conserved region of yeast Cox18 in E. coli, we show here that Cox18 is able to complement the essential Sec-independent function of YidC. This identifies Cox18 as a bona fide member of the. approach in E. coli. The data demonstrate that Cox18 complements the essential Sec-independent function of YidC. Hence, by this criterion, Cox18 can be considered a bona fide member of the YidC ⁄ Oxa1. conserved in YidC and Cox18. To study the functioning of Cox18 in E. coli, a hybrid protein was constructed consisting of the first 247 amino acids of YidC fused to the mature part of yeast Cox18 (residues