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, mitoch
Trang 1the essential Sec-independent function of
Escherichia coli YidC
Edwin van Bloois1, Gregory Koningstein1, Heike Bauerschmitt2,3, Johannes M Herrmann2and Joen Luirink1
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¨t Mu¨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.
Trang 2Pull-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 Foc (subunit
of the F1F0-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 Ec-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
Trang 3expressed 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 mid pEH1, or the empty vector or EcOxa1 in the expression plas-mid pEH1 (as controls), was grown to plas-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.
Trang 4IPTG, 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 F1F0-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 l M
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).
Trang 5insertion 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, asbiogene-sisting 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 [35S]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 D660units 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.
Trang 6IMVs 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 posirepresenta-tion 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-tRNAsup 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.
Trang 7cross-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 Foc
subunit of the F1F0-ATPase and of the N-terminus of
CyoA (see above) is exclusively dependent on YidC
Consequently, F1F0-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 Foc 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
Trang 8[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-LightPLUS western blotting substrate were
obtained from Roche Molecular Biochemicals (Mannheim,
Germany) [35S]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 colistrain 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-tRNAsup
[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
[35,42,49] For in vitro photo-cross-linking experiments,
pC4Meth50LepTAG10 and pC4Meth50LepTAG15 were
used [6,13] Plasmid 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
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.Km and pCL-EcOxa1.Sm [35] with the KpnI⁄ SmaI fragment of pEH1-EcCox18His, yielding pCL-EcCox18.Km and pCL-EcCox18.Sm The nucleotide sequences of all constructs were verified by DNA sequencing
In vivo processing assays
JS7131 harbouring pASKIBA3-CyoAHA and pCL1921Km,
or pCL-EcCox18.Km, 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 pCL1921Km, or pCL-Ec-Cox18.Km, 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
Trang 9Samuel 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
Trang 10prokaryotes 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
cerevisiaeCox2p 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-terN-ter-minal 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-siaemitochondria 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