As the first cytochrome bc1 structures were characterized, two fea-tures of the ISP came as a major surprise: a the ISP intertwines between the monomeric halves of the enzyme, such that t
Trang 1inserted into the cytoplasmic membrane by the
twin-arginine translocase
Julie Bachmann1, Brigitte Bauer1, Klaus Zwicker2, Bernd Ludwig1and Oliver Anderka1,*
1 Institut fu¨r Biochemie, Johann Wolfgang Goethe-Universita¨t, Frankfurt, Germany
2 Zentrum der Biologischen Chemie, Institut fu¨r Molekulare Bioenergetik, Universita¨ts-Klinikum, Frankfurt, Germany
Mitochondrial respiratory complex III⁄ cytochrome bc1
is among the best-characterized membrane proteins,
with structures elucidated from several species [1–4]
These structures revealed the organization of three
cat-alytic subunits (SU) in the homodimeric complex; these
are cytochrome b, cytochrome c1, and the Rieske iron–
sulfur protein (ISP) Cytochrome b forms eight
trans-membrane (TM) helices that bind two hemes and is
largely contained within the membrane bilayer Both
cytochrome c1 and the ISP are single-spanning TM proteins with globular hydrophilic domains located in the periplasmic space; these ecto-domains carry the heme and [2Fe)2S] cofactors, respectively As the first cytochrome bc1 structures were characterized, two fea-tures of the ISP came as a major surprise: (a) the ISP intertwines between the monomeric halves of the enzyme, such that the N-terminal TM helix of a given ISP is anchored within one monomer, whereas the
Keywords
cytochrome bc 1 complex; membrane
targeting; Paracoccus denitrificans;
Rieske iron–sulfur protein; twin-arginine
translocation
Correspondence
O Anderka, Institut fu¨r Biochemie,
Johann Wolfgang Goethe-Universita¨t,
D-60438 Frankfurt, Germany
Fax: +49 69 3058 1901
Tel: +49 69 3051 2418
E-mail: oliver.anderka@sanofi-aventis.com
*Present address
Sanofi-aventis, TD Metabolism, Frankfurt,
Germany
(Received 16 June 2006, revised 23 August
2006, accepted 24 August 2006)
doi:10.1111/j.1742-4658.2006.05480.x
The Rieske [2Fe)2S] protein (ISP) is an essential subunit of cyto-chrome bc1 complexes in mitochondrial and bacterial respiratory chains Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway Here, we provide experimental evidence that membrane integra-tion of the bacterial ISP indeed relies on the Tat translocon We show that targeting of the ISP depends on the twin-arginine motif A strict require-ment is established particularly for the second arginine residue (R16); con-servative replacement of the first arginine (R15K) still permits substantial ISP transport Comparative sequence analysis reveals characteristics com-mon to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved ‘+5’-consensus motif lysine residue Substitution of the +5 lysine (Y20K) compromises ISP export and⁄ or cytochrome bc1 stability to some extent and points to a specific role for this deviation from the canonical Tat motif EPR spectroscopy confirms cytosolic insertion of the [2Fe)2S] cofactor Mutation of an essential cofac-tor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway
Abbreviations
EPR, electron paramagnetic resonance spectroscopy; ISF, Rieske iron–sulfur protein soluble fragment; ISP, Rieske iron–sulfur protein;
SU, subunit; Tat, twin-arginine translocation; TM, transmembrane.
Trang 2periplasmic domain structurally and functionally
inter-acts with the other monomer; (b) the periplasmic
domain seems to undergo large-scale motion in order
to shuffle electrons between cytochromes b and c1 Up
to eight accessory subunits surround the catalytic core
of the enzyme; they are probably required for assembly
and⁄ or stability of the complex, but their precise
func-tion is largely unknown
Cytochrome bc1 complexes from bacterial
respirat-ory chains, e.g from Paracoccus denitificans, are made
up of only the catalytically essential subunits, and
show high sequence identity towards their
mitochond-rial counterparts [5,6] There is considerable interest in
studying these minimal complexes as model systems;
they are readily amenable to genetic manipulation and
therefore allow unsolved issues of mechanism or
bio-genesis to be tackled However, structures of such
‘minimal’ bc1 complexes cannot currently be solved to
high resolution In the case of the related b6f complex
of oxygenic photosynthesis, a structure of prokaryotic
origin has recently been characterized [7]
Despite its relatively simple composition, there is
currently little information about biogenesis and
assembly of the prokaryotic bc1 complex It is not
known how cytochrome b as the central and largest
subunit is inserted into the membrane The
cyto-chrome c1 precursor is translocated along the Sec
translocon; its heme cofactor is exported to the
peri-plasm and attached to the apo-protein by the c-type
cytochrome maturation machinery [8,9] A
twin-argin-ine-dependent translocation (Tat) was first proposed
for the Rieske iron–sulfur protein by Berks [10], based
on the occurrence of a specific consensus motif in its
N-terminal region Since then, considerable
informa-tion has been obtained about the Tat system [11–13]
Its hallmarks are: (a) the occurrence of and export
dependence on a S⁄ T-R-R-x-F-L-K consensus motif
within a tripartite signal peptide; (b) proton-motive
force-dependent and ATP-independent transport; (c)
insertion of cofactors and⁄ or assembly of different
subunits at a cytosolic stage; and (d) export in a fully
folded conformation, which is probably the most
remarkable feature Components of the Tat
trans-locon are TM proteins TatA, B, and C TatBC seems
to form the initial receptor [14] Electron microscopy
reveals that multiple copies of TatA form ring-like
structures which are thought to represent the
translo-cation pore [15] Recently, specific chaperones have
been identified which seem to exert ‘proof-reading’ or
‘quality control’ on the Tat translocon substrates
[16,17] In thylakoids, a ‘DpH pathway’ has been
des-cribed that is homologous to the bacterial Tat pathway
[18]
Currently known Tat substrates are almost exclu-sively soluble periplasmic proteins; to date only five Escherichia coli proteins containing a C-terminal mem-brane anchor have been shown to be transported along the Tat pathway In contrast, the Rieske ISP is N-ter-minally anchored, which is novel and unique for a putative Tat substrate: The N-terminus would serve a dual role of export signal and membrane anchor In the thylakoid system, it has been already shown that the ISP is transported via the DpH⁄ Tat pathway [19,20] Interestingly, the chloroplast ISP displays a
KR motif, which is only the second known example
of natural deviation from the otherwise invariant
RR motif [21]
We examined membrane translocation of the ISP from P denitrificans Experimental evidence is provi-ded that the bacterial ISP is indeed a substrate of the Tat translocon, and transport depends on the presence
of the Tat consensus motif However, as in the case of the thylakoid ISP and in contrast to the majority of other Tat substrates, transport of the P denitrificans ISP shows more relaxed requirements regarding the conserved RR motif Furthermore, bioinformatic ana-lysis and site-directed mutagenesis reveal distinctive features of a Tat signal that simultaneously serves as a membrane anchor
Results
Bacterial Rieske proteins contain signal sequences that deviate from the canonical Tat consensus
In an early review on double-arginine signal sequences, the ISP of P denitrificans was listed as a potential sub-strate for what was later named the Tat pathway [10]
To substantiate this assignment, the P denitrificans ISP was initially analysed using bioinformatic tools Its primary sequence was aligned to ISP sequences from other proteobacteria Sequences were selected accord-ing to a phylogenetic study on Rieske proteins [22] All chosen ISPs are subunits of respiratory cytochrome bc1 complexes The main part of the sequence representing the cluster-binding periplasmic domain was omitted from the comparison to avoid biasing the alignment towards this highly conserved protein region, which might mask the similarities of interest within the N-terminal part
The alignment shown in Fig 1 reveals that all selec-ted sequences contain the indicative twin-arginine Comparison with the canonical Tat consensus (S⁄ T)-R-R-x-F-L-K [10] shows good agreement in the other positions of the motif, with the remarkable exception
Trang 3of the C-terminal lysine residue, which is not found in
any of the ISP sequences examined On average, a
lysine residue appears in this position in > 60% of
general Tat signal sequences [10] This position is
num-bered ‘+5’, relative to the first invariant arginine; it
corresponds to Y20 in the P denitrificans sequence
and is discussed in more detail later Upstream of the
consensus motif, a mean of 11 residues is found,
con-sistent with the frequently observed extended n-region
of Tat signal sequences relative to Sec signals [23] The
upstream sequences do not exhibit sequence
conserva-tion; in contrast, it has been observed that signal
pep-tides for proteins binding a given cofactor (e.g the
[Ni–Fe] hydrogenase small subunits) often show
marked sequence conservation within this region [11]
For the n-region of cofactor-containing Tat substrates,
an a-helical structure was proposed [12] Using jpred
(see Experimental procedures), a corresponding
secon-dary structure could not be predicted for bacterial
Rieske proteins (data not shown) The h-region was
defined according to a set of rules given by Cristobal
et al [23]; it consists of 19 residues, in good agreement
with a length of 15–20 residues found within known
Tat signals It is in remarkable contrast to established
Tat substrate proteins that a number of conserved
resi-dues can be found within the ISP h-region (for
discus-sion, see below) The c-region of the putative ISP Tat
signal predominantly displays an initial proline residue
which has been described for other Tat signals as a
helix breaker following the a-helical h-region [23]
However, Tat signal peptides characteristically contain
basic amino acids within the h-region that serve as a
‘Sec-avoidance signal’; these basic residues are not
observed in the analysed ISP sequences All Rieske
sequences lack the AxA cleavage site at the end of the
c-region for obvious reasons, as this part of the ISP serves as a membrane anchor The c-region overlaps with the flexible hinge-region observed in the crystal structures of the mitochondrial enzyme which allows for movement of the Rieske ecto-domain within the cytochrome bc1 complex [1,4,24] Taken together, the N-terminal domain of Rieske proteins displays several hallmarks of Tat signal peptides, such as the invariant twin-arginine and the tripartite structure However, it also deviates in important aspects, missing for example the consensus lysine or a ‘Sec-avoidance’ signal The N-terminal part of Rieske proteins serves as a membrane anchor, whereas the majority of known Tat substrates are exported to the periplasm where their export signals are cleaved In order to examine this difference in detail, the corresponding h-regions were compared Kyte–Doolittle analysis was performed with
a sequence window size of 19, appropriate for detect-ing potential TM helices [25] ISP sequences were selec-ted according to the sequence alignment in Fig 1 For known Tat substrates, used as a comparison group, three to five sequences were taken from five different classes each: [NiFe] hydrogenase small subunits, MauM family ferredoxins, NapA periplasmic nitrate reductases, NosZ nitrous oxide reductases, and TorA Trimethylamine-N-oxide reductases (for details, see Experimental procedures) The resulting Kyte–Doolit-tle data were aligned relative to the Tat consensus motif An averaged hydropathy index was calculated for the ISP sequences and the comparison group (Fig 2) Both curves display a positive score in the h-region which corresponds to relative hydrophobicity The data show that the ISP group is distinctly more hydrophobic than the comparison group; this differ-ence is statistically significant (P < 0.001, two-tailed
Fig 1 Bacterial ISP sequences contain the twin-arginine consensus specific of the Tat translocation pathway Sequence alignment of Rieske proteins that are subunits of proteobacterial cytochrome bc 1 complexes The C-terminal portion of the sequences representing the cluster-binding hydrophilic domain is removed to avoid the alignment being biased towards this highly conserved protein region Sequences were retrieved from the SwissProt server and the alignment performed with CLUSTAL X , as detailed in the Experimental procedures Star symbols denote invariant residues, colons highly conserved and dots conserved positions Limits of the h-region were determined following rules given previously [23] The start of the so-called hinge region is indicated [4,57] (Lower) Canonical Tat consensus motif.
Trang 4Mann–Whitney U-test with pooled data for the
corres-ponding h-regions) However, the ISP group h-region
shows relatively weak hydrophobicity with hydropathy
values < 1.5 compared with TM helices of
multispan-ning membrane proteins which typically reach
hydro-pathy values of > 1.8 in the Kyte–Doolittle analysis,
using a window size of 19 [25] This was confirmed
with a small selection of single-spanning membrane
proteins from P denitrificans; here, hydropathy values
for the TM helices ranged between 2 and 3 (data not
shown) It was observed that the h-region of Sec signal
peptides is significantly more hydrophobic than the
h-region of common Tat peptides [23]; this also holds
true when the ISP signal sequence is compared with
Sec signal peptides For a set of 20 predicted Sec
sub-strates from P denitrificans, a mean hydropathy value
of 1.8 (± 0.1 SEM) was obtained for the h-region;
the set of ISP h-regions showed a mean hydropathy
value of only 1.0 (± 0.1 SEM) in the Kyte–Doolittle
analysis (details not shown; here, a window size of 9
was applied to both data sets)
To obtain comparative information from a different
method, TM helix prediction was performed using the
program tmap [26] As an input, multiple sequence
alignments were used that were generated with
clu-stal x [27] The algorithm predicts a TM helix for the
ISP group only, not for the five different classes of Tat
substrates mentioned above Most remarkably,
predic-tion of the ISP TM helix was absolutely dependent on
the natural deviation from the canonical consensus motif described above: When a ‘+5’ lysine residue of the Tat consensus was introduced in silico (e.g a Y20K mutation in the P denitrificans sequence, see also below), TM prediction failed in all examined ISP sequences In conclusion, slightly higher mean hydro-phobicity compared with average Tat signal peptides and the exchange of the canonical ‘+5’ lysine residue against a more hydrophobic amino acid (isoleucine, phenylalanine, tyrosine) provide a clear discrimination and an initial evidence for the ISP signal sequence to simultaneously serve as a membrane anchor
Finally, to predict whether the Tat translocation machinery is operating in P denitrificans, the draft version of the genome was inspected (Joint Genome Institute Microbial Sequencing Program) Three genes annotated as TatA, TatB, and TatC homologues could be found on contig 67; TatB and TatC are adjacent genes and might form a transcriptional unit, whereas the TatA homologue is found in a separate locus
Specific mutations demonstrate membrane insertion of the P denitrificans Rieske protein via the Tat pathway
In order to analyse membrane insertion of the ISP in
P denitrificans, a number of mutants was generated Individually and in combination, the invariant arginine residues of the consensus motif were conservatively exchanged for lysine In addition, a Y20K mutation introduces the ‘+5’ lysine residue that is ‘missing’ in the ISP sequences As export via the Tat pathway clas-sically requires previous cofactor-insertion in the cyto-plasm, a mutation C152S was introduced that conservatively replaces one of the cluster-binding lig-ands Site-directed mutagenesis and cloning procedures were performed as described in the Experimental pro-cedures, and mutations were confirmed by sequencing Wild-type and ISP mutants of the complete fbc operon coding for the three-subunit cytochrome bc1 complex under control of its native promotor were cloned into
a broad host-range vector and introduced into a
P denitrificans Dfbc::Km strain [28] via conjugation Expression of the ISP subunit was probed by western blotting of whole-cell samples (not shown)
For subcellular fractionation of P denitrificans cells,
a protocol originally developed for E coli was adapted (Experimental procedures) To check the effectiveness
of the process, three markers characteristic for each subcellular fraction were assayed (Table 1) Redox-difference spectra were recorded and the amount of soluble c-type cytochromes determined, these are
Fig 2 The signal sequence h-region in general Tat substrates is
significantly less hydrophobic compared with Rieske proteins.
Kyte–Doolittle plot comparing the hydropathy of ISPs and general
Tat substrate proteins Values fall within a range of +4 to )4, with
hydrophilic residues having a negative score Each data point
represents an averaged hydropathy value derived from analysis of
multiple sequences, as detailed in the Experimental procedures.
A relative sequence numbering is given, with position 0
represent-ing the first invariant arginine residue of the consensus motif.
Boundaries of the h-region are indicated as defined in Fig 1.
Trang 5normally found only in the periplasm, but not in the
cytosolic fraction [29] Enzymatic activities of cytosolic
malate dehydrogenase and membrane-bound
cyto-chrome c oxidase were the two other markers that
allowed any cross-contamination to be assessed The
periplasm was isolated efficiently, as demonstrated by
the high relative yield of c-type cytochromes given in
Table 1 Within the periplasmic fraction, no malate
dehydrogenase activity was detectable; this confirms
that practically no cell lysis occurred during extraction
of the periplasm The enzymatic activites of malate
dehydrogenase and cytochrome c oxidase show that
there is little cross-contamination between the cytosolic
and membrane fractions However, good separation,
as demonstrated here, could be achieved only after
repeated ultracentrifugation Small differences between
the total activity in the nonfractionated cell lysate and
the sum of the individual fractions can be easily
explained by either loss of material or protein
degrada-tion during the procedure Taken together, the
sub-cellular fractionation method applied here resulted in
essentially quantitative separation with
cross-contamin-ation of a few per cent at most
Localization of the ISP variants was analysed by
western blotting of subcellular fractions derived from
small-scale cultures of P denitrificans in the
exponen-tial growth phase (50 mL, D600 1.5) The result of
this experiment is given in Fig 3 The deletion strain with the wild-type protein expressed in trans showed a dominant ISP signal in the membrane fraction; how-ever, substantial amounts of the protein could be found in the cytoplasm It should be mentioned that the Rieske protein typically separates into two bands
on SDS⁄ PAGE; the lower band can be seen here only
in the fractions with elevated ISP amounts Exchange
of the first invariant arginine (R15K) leads to a com-parable distribution, but the total ISP level is clearly diminished In contrast, both the R16K mutation and the R15K⁄ R16K double mutation result in almost complete loss of the signal in the membrane fraction The bands for the Y20K mutant resemble the wild-type, with slightly decreased levels A much weaker sig-nal was obtained for the C152S mutant, which should abolish cofactor binding This is in line with earlier observations by Davidson et al [30]; they prepared an equivalent mutation in the closely related Rhodobacter capsulatus complex and observed a strongly decreased membrane level of the apo-ISP that was between one and two orders of magnitude less than the wild-type overproducer Interestingly, a decrease or loss of the membrane-bound form in the mutant strains was not accompanied by ISP accumulation in the cytosol, pointing to rapid degradation of ISP that cannot be targeted to the membrane
Table 1 P denitrificans cells are efficiently fractionated Cell cultures at exponential growth (D 600 )1.5) were fractionated as described in the Experimental procedures For each cell fraction, a marker protein was assayed Periplasmically located c-type cytochromes were quantified using redox difference spectra Activities of the cytosolic malate dehydrogenase and the membrane-integral cytochrome c oxidase were assayed as described in the Experimental procedures, and the total activities of the cell fractions were compared Three separate fractiona-tions gave consistent results; values given here are from a single representative experiment ND, values not determined.
Marker
Cell fraction (%)
Fig 3 Specific mutations strongly inhibit membrane insertion of the P denitrificans Rieske protein Detection of ISP by western blotting in cell fractions from different P denitrificans strains Strain variants are indicated above the corresponding lanes, as follows: wt, P denitrifi-cans bc1deletion strain MK6 expressing the wild-type fbc operon from plasmid pAN42; R15K, R16K, R15 ⁄ R16K, Y20K, and C152S, MK6 strain bearing pAN42 derivatives with the respective mutation(s) in the fbcF gene Total amounts of 15 lg protein were loaded in each lane The applied cell fractions are indicated: C, cytoplasm; M, membrane; periplasmic samples did not show any ISP signal and therefore are omitted The Rieske protein typically separates into two bands on SDS ⁄ PAGE, as indicated by the two arrows; the lower band shows up only with higher protein amounts.
Trang 6Cytochrome bc1 enzymatic activity was assayed to
obtain a more quantitative measure of decreased ISP
amounts in the mutant membranes To this end, we
assume that mutations in the signal sequence do not
exert an effect on the intrinsic activity of the enzyme;
this seems plausible as the structures of mitochondrial
homologues show that the N-terminal part of the ISP
is far from the catalytic centres and is separated by the
membrane dielectric in the native environment [3,4]
Obviously, this argument does not apply for the
C152S mutant Specific QH2:cytochrome c
oxidoreduc-tase activities of membrane samples are given in
Table 2 The data show that mutant membranes R16K
and R15K⁄ R16K were essentially inactive, whereas the
R15K and the Y20K mutant membranes contained
considerable amounts of fully assembled and active
enzyme As expected, the C152S mutant was fully
inac-tive For the Y20K mutant, an interesting observation
was made when the membranes were subjected to
sodium carbonate treatment prior to activity
measure-ments Although this treatment had only a minor
effect on wild-type membranes, pretreated Y20K
mem-branes showed severe instability when recording
steady-state activities spectroscopically: traces with
ini-tially normal slopes became a flat line a few seconds
after the addition of substrate (data not shown)
In conclusion, enzymatic data from Table 2 and the
western blot results given in Fig 3 provide a consistent
picture with strong evidence for a Tat-dependent
trans-location of the P denitrificans ISP Conservative
exchanges of the twin-arginine motif for a lysine pair
blocked membrane insertion; the single mutations on
the arginine pair showed differential effects, with a
more important role for the second arginine residue
As anticipated by the above sequence analysis, ‘restor-ation’ of the canonical Tat consensus with the Y20K mutant has a negative impact on membrane insertion; somewhat surprisingly, this effect is rather mild, and
to a considerable extent the mutant ISP is still func-tionally incorporated into the cytoplasmic membrane
In addition, the effect of sodium carbonate treatment points to a structurally destabilizing effect of this mutation Finally, removal of cofactor binding capa-city with the C152S mutant strongly impairs mem-brane insertion; this result might be explained by the postulated ‘cofactor-proof-reading’ operating in the Tat translocation scheme [11,31] However, certain amounts of the apo-ISP are still found in the mem-brane; there is obviously no strict requirement for cofactor insertion prior to transport Furthermore, a potential drawback of our data is that the effects of site-specific mutants are deduced only from the steady-state distribution of the ISP; no kinetic data for mem-brane translocation were obtained Particularly in case
of the C152S cofactor insertion mutant and the Y20K mutation in the ‘+5’ position, it is conceivable that secondary effects such as proteolytic degradation due
to improper folding or an assembly defect of the
bc1 complex with subsequent proteolysis might also account for the reduced amounts of ISP in the mem-brane Further experiments will be needed to fully exclude such side effects
The [2Fe)2S] cluster of the Rieske protein is inserted in the cytoplasm
As cytosolic cofactor insertion is one of the hallmarks
of Tat translocation, the cofactor loading status of the ISP in P denitrificans cytosolic fractions was examined using electron paramagnetic resonance (EPR) Cells from a 0.5 L culture in the exponential growth phase were harvested; cytosolic fractions of the complemented wild-type, the export mutant R15K⁄ R16K, and the cofactor binding mutant C152S were isolated and concentrated by ultrafiltration Membranes from the complemented wild-type and the C152S mutant were used as positive and negative controls for the presence of the Rieske [2Fe)2S] clus-ter EPR signal A reference spectrum was recorded with a purified sample of the Rieske protein fragment (ISF) EPR samples were reduced with 5 mm sodium ascorbate (Fig 4)
Spectrum I shows the reference spectrum of the purified ISF; the complemented wild-type in spec-trum II gave a clear Rieske signal in the membrane fraction, with shifted peak positions relative to
Table 2 Cytochrome bc 1 activity reflects the decreased ISP
con-tent in mutant membranes Membranes were isolated from the
parental fbc::Km deletion strain that was complemented with a
wild-type copy of the fbc operon in trans or mutants thereof
Val-ues given are the average of three to five measurements To
elim-inate unspecific activity effects, activity data were corrected for the
slope measured when the enzyme was inhibited with 10 l M
anti-mycin A Relative activity refers to the wild-type complemented
strain.
Strain
Specific activity of membrane fraction (mUÆmg)1)
Relative activity (%)
Trang 7spectrum I This shift most probably arises from
h-bond interactions of a histidine cluster ligand with
the quinone substrate bound to the membrane integral
cytochrome bc1 complex [32] No [2Fe)2S] cluster
sig-nal was visible in the cytosolic and membrane fractions
of the C152S mutant, demonstrating: (a) the inability
of the mutant to insert a cofactor, and (b) the specific
origin of the signal in the other samples The cytosolic
fraction of the complemented wild-type clearly showed
the EPR signature of the Rieske cluster (spectrum V),
which provides strong evidence for the cytosolic
assem-bly of the holoprotein Likewise, the cytosol of the
R15K⁄ R16K double mutant contained the Rieske
clus-ter, albeit at lower concentration compared with the
wild-type cytosol (spectrum VI) In order to
demon-strate the presence of the cluster in this strain more
convincingly, the cytosolic fraction obtained from a
2.5 L culture was further enriched It was applied to a
Q Sepharose column, and the 150–250 mm NaCl salt
gradient eluate was pooled and concentrated; the ISP
is known to elute at 200 mm NaCl [33] The
enriched cytosolic fraction clearly shows the indicative
Rieske spectrum (VII) The existence of the Rieske
cluster in the cytosol of the R15K⁄ R16K which is
incompetent of membrane insertion (Fig 3) clearly
rules out the possibility that the signal could arise
from membrane remnants in the cytosolic fraction
Furthermore, no Rieske cluster signal was observed in
membrane samples of the R15K⁄ R16K double mutant
(not shown) Thus, the EPR results clearly
demon-strate cytosolic cofactor insertion which is a typical
feature of Tat substrate proteins
Discussion
The aim of this study was to obtain experimental evi-dence of whether the Rieske [2Fe)2S] protein subunit
of bacterial cytochrome bc1 complexes is targeted to the cytoplasmic membrane by means of twin-arginine-dependent translocation Studying the ISP from
P denitrificans with sequence analysis tools, site-direc-ted mutagenesis, and EPR spectroscopy, we found the key requirements of Tat translocation fulfilled: the characteristic features of the signal sequence, the export dependence on the conserved arginine pair,
Fig 4 The [2Fe )2S] cluster is inserted into the Rieske apoprotein
in the cytoplasm EPR spectra of: I, purified Rieske protein
frag-ment (ISF); II, membranes of complefrag-mented wild-type strain; III,
membranes of C152S mutant; IV, cytosol of C152 mutant; V,
cyto-sol of complemented wild-type; VI, cytocyto-sol of R15K ⁄ R16K double
mutant; VII, IEX chromatography-enriched cytosol fraction of
R15K ⁄ R16K double mutant (150–250 m M NaCl eluate) Samples
were reduced with 5 m M sodium ascorbate For representation
pur-poses, spectra are scaled differently on the y-axis: Spectra II–VII
are magnified by a scaling factor of 500 relative to spectrum I This
scaling factor includes differences in sample concentration, spectral
accumulation, and graphical scaling Hence, spectral intensities do
not reflect the concentration ratio between membrane and cytosol
fractions Peaks in the g x and g y region are indicated by vertical
lines; the g z region is omitted due to overlaps with EPR signals
from other proteins The positions of gxsignals in samples I and VII
(g x1 ) are shifted to higher magnetic field because of the occurrence
of ligand-free iron–sulfur protein Conditions for EPR spectroscopy
are given in the Experimental procedures.
Trang 8and cytosolic cofactor insertion as a prerequisite for
membrane targeting
To our knowledge, the only experimental evidence
for Tat dependence of the bacterial ISP to date is the
finding that a DtatBC deletion mutant of R
leguminos-arum lacks a functional bc1 complex [34] In contrast,
insertion of the chloroplast ISP into the thylakoidal
membrane via the Tat⁄ DpH-pathway is well
documen-ted [19,20] This protein was the first Tat substrate
shown to be an integral membrane polypeptide with a
signal sequence that is not cleaved after translocation
Another interesting feature is the lack of the ‘invariant’
twin-arginine; instead, a KR sequence is found in the
corresponding position In contrast, cyanobacteria as
supposed ancestors of chlorplasts contain ISPs with a
perfect twin-arginine motif It was argued that the RR
to KR transition has a functional role in slowing
import of the now nucleus-encoded ISP to allow for
proper cofactor insertion in the stroma [19]
Associ-ation of the ISP with stromal chaperonin Cpn60
and⁄ or Hsp70 was observed [19,35] Furthermore,
evi-dence was found for interplay with components of the
Sec system and it was hypothesized that the ISP is an
‘intermediate’ substrate in the evolution of the
chloro-plast export pathways [19]
The second conserved arginine residue plays a
critical role in ISP translocation
The relative amounts of ISP, detected by
immunologi-cal means in the membrane fractions of the variant
strains R15K, R16K, and R15K⁄ R16K, show that
both arginine residues are important for membrane
targeting However, the second arginine appears the
most critical, and even a conservative mutation in this
position leads to an essentially complete block,
whereas replacement of the first arginine allows
sub-stantial membrane insertion This is a remarkable
find-ing in the light of the naturally occurrfind-ing KR motif
in plant ISPs This observation raises the question
whether the Tat translocon is especially ‘permissive’
towards the Rieske protein, allowing for variation at
the first arginine position, or whether the stricter role
of the second arginine is a general feature of Tat
sub-strates Originally, an absolute requirement for both
arginines was stated [11,36,37] A gain-of-function
mutant screen with a Tat-targeted GFP reporter
con-struct, however, indicated that both positions tolerate
variation, with the second position even being more
flexible Similarly, the E coli multi-copper oxidase
superfamily homologue SufI allowed single
conserva-tive substitutions at both positions In contrast to this,
several authors report a critical role only for the
second arginine [21,38–40] Furthermore, apart from the plant ISPs, another example of natural ‘KR’ vari-ation of the Tat motif is known, interestingly, also in case of a protein carrying an iron–sulfur cofactor [21] Taken together, these examples show that in some sig-nal peptides at least, conservative variation especially
in the first position of the twin-arginine is possible, and this idea is corroborated by this study
The ISP Tat signal serves as a membrane anchor Sequence analysis of the N-terminal ISP portion is in good overall agreement with the structure of general Tat signal peptides However, it shows some clear dis-tinctions that may account for its dual role as a Tat signal and as a membrane anchor The ISP h-region exhibits a significantly higher hydrophobicity than average Tat signal peptides (Fig 2) Likewise, it is well established that Sec signal peptides show higher hydro-phobicity than typical Tat signals An engineered increase in hydrophobicity in a Tat signal peptide even leads to a (nonphysiological and functionally inexpedi-ent) re-routing of a precursor protein to the Sec trans-locon [23] We wondered if the ISP h-region has a similar degree of hydrophobicity as the corresponding portion of Sec signal peptides However, when we compared the h-region hydropathy values of the ISPs and a set of Sec substrates, we found the ISP h-region
to be considerably less hydrophobic (not shown) Therefore, a clear ranking of hydropathy values becomes apparent, with Sec h-regions as the most hydrophobic, followed by ISP signal h-regions, which again are distinctly more hydrophobic than generic Tat h-regions Together with the fact that we do not find a basic ‘Sec-avoidance’ signal [41] in the c-region, the elevated hydrophobicity of the h-region raises the intriguing question whether the bacterial ISP may also interact with Sec components Evidence for such inter-play exists in case of the chloroplast ISP, where it was suggested that soluble components involved in Sec-targeting also deliver the Rieske protein to the Tat translocon [19] It will be interesting to see if future experiments show a similar linkage in case of the bacterial ISP
As another critical determinant for membrane anchorage, the moderately hydrophobic residue at the
‘+5’ position was identified in silico, which was found
in place of the consensus lysine in the ISP Tat motifs Genetic substitution by the canonical lysine residue leads to slightly decreased ISP levels in the cytoplasmic membrane This could be interpreted in line with the observation made by Stanley et al [42] that a ‘+5’ lysine slows export of Tat substrates Alternatively,
Trang 9our results may be explained by secondary effects of
the Y20K mutation which might destabilize the bc1
complex and lead to proteolysis At any rate, the
hypothesis of these authors that this slowing has the
physiological role of allowing for proper cofactor
insertion is not substantiated here; the ISP is a
cofac-tor-containing protein but lacks the ‘+5’ lysine in its
native sequence Probably, retardation is needed for
other cofactor classes or in the case of heterodimeric
proteins, where the ‘hitch-hiking’ subunit is granted
time to associate with the subunit containing the Tat
signal peptide [12]
Activity measurements with Y20K mutant
mem-branes pretreated with carbonate show apparent rapid
loss of cytochrome bc1 activity; this can be tentatively
interpreted as a less stable insertion of the ISP into the
hydrophobic core of the enzyme complex It has been
a frequent observation that the Rieske subunit appears
only poorly associated with the bc1 complex, is easily
lost during purification of the bc1 complex, and can be
extracted by high detergent concentrations or
chao-tropic salt treatment [43]; before the crystal structure
information emerged, it was still a matter of debate
whether the ISP is a true integral membrane protein
[44,45] This weak association may be explained by the
h-region hydrophobicity as given in Fig 2, which,
albeit being higher as for the Tat substrate average, is
still rather low compared with TM helices of other
membrane-anchored proteins Possibly, the lacking
‘+5’ lysine, on the one hand, and the comparatively
low hydrophobicity, on the other hand, represent a
compromise between the conflicting requirements of
TM helix formation and acceptance as a substrate by
the Tat translocon
Recently, the crystal structure of a cytochrome bc1
homologue, the cytochrome b6f complex from the
cyanobacterium Mastigocladus laminosus was solved
[7] As discussed by Berks et al [13], this enzyme also
contains an ISP subunit with a putative Tat signal
Here, an asparagine residue is in the ‘+5’-position;
sequence alignments of ISP subunits from bacterial b6f
complexes show that for this subgroup asparagine is
the most frequent amino acid in this position (data not
shown), whereas the canonical lysine residue cannot be
found, as in the case of bacterial bc1 complexes In the
enzyme structure, no major interactions were found
for the ‘+5’-Asn side chain However, whereas the
invariant Arg residues are located at the membrane–
water interface, the ‘+5’-residue lies well within the
TM region of the enzyme It is a reasonable
assump-tion that a Lys residue cannot be accommodated in
this hydropohobic environment and is therefore absent
from the Tat motif of bacterial ISPs
Potential steps during ISP biogenesis The observation that cofactor-containing periplasmic proteins carry a conserved twin-arginine motif led to discovery of the Tat translocation pathway [10] Cyto-solic cofactor insertion is a key feature of Tat sub-strates and was experimentally confirmed by a number
of studies for the bacterial and the homologous thylak-oid system, as reviewed by Berks et al [11] Our EPR data demonstrate the presence of holo-ISP in cytosolic fractions, thereby confirming that cluster assembly takes place in the cytosol Impaired membrane inser-tion in the C152S mutant may indicate that cofactor insertion is a prerequisite for efficient export of the ISP It was convincingly shown by different authors that a lack of the cofactor prevents export of Tat sub-strates [31,36] However, because the kinetics of mem-brane insertion were not examined experimentally, the possibility exists that the apo-ISP is targeted to the membrane perfectly normally and only secondary pro-teolysis depletes the membrane fraction It is an inter-esting observation that the cluster content of the R15K⁄ R16K mutant cytosol was much lower than in the complemented wild-type (Fig 4); for an export-deficient strain, rather an accumulation of the signal might be expected It is therefore tempting to speculate
on a potential interplay of the cluster insertion machinery and the Tat translocation process With a closer look at the spectra in Fig 4, it is remarkable to see that the chromatographically enriched cytosol frac-tion of R15K⁄ R16K (spectrum VII) shows the same g-value positions as the purified reference protein (I), whereas cytosolic fractions taken directly for EPR measurements (spectra V + VI) resemble spectrum II from intact bc1 complex where the [2Fe)2S] cluster histidine ligand is involved in hydrogen bonding inter-actions Interaction with a quinone molecule in the cytosol appears quite unlikely; therefore, this observa-tion may give a hint to a putative binding parter of the ISP in the cytosol, probably playing some chaper-one role The chromatographic purification step may have removed this binding partner Further experi-ments are needed to examine this aspect in detail
A potential binding partner could be involved in cluster assembly; likely candidates are components of the Nif
or Isc machinery responsible for iron–sulfur cluster assembly in bacteria and mitochondria [46,47] Prelim-inary data from our laboratory indicate that overex-pression of the isc or nif operon can indeed promote [2Fe)2S] cluster assembly to the P denitrificans ISP in the heterologous host E coli [48] In a BLAST search
on the draft version of the P denitrificans genome, putative genes homologous to those of the isc operon
Trang 10from E coli and the nif operon from A vinelandii were
identified (data not shown)
Alternatively, ‘proof-reading’ chaperones may
inter-act with the ISP in the cytosol Recent evidence
points to such specific chaperones acting on Tat
sub-strates and preventing premature translocation [16,49]
However, such specific proof-reading chaperones were
typically acknowledged as accessory genes in the
operon context of the respective Tat substrate protein
[40] No such ORF of yet unknown function is
pre-sent in the fbc operon coding for the cytochrome bc1
complex of P denitrificans Also, chaperone binding is
assumed to be associated with conserved sequence
ele-ments in the signal peptide n-region [12] We did not
find such sequence conservation in case of bacterial
ISPs; however, comparison of h-regions shows
signifi-cant similarities among the different ISPs and might
therefore be specifically recognized by a putative
chaperone
An additional level of control for export competence
is designated ‘quality control’ [17] Here, the folding
status of the protein is examined, presumably by the
Tat translocon itself From our data it is not clear
whether cofactor ‘proof-reading’ or general
‘quality-control’ keeps the apo-ISP from being efficiently
trans-located and inserted into the membrane; a third
explanation for the lower membrane levels, as
men-tioned above, is that the apo-ISP is normally targeted
but subsequently degraded by periplasmic proteases
From the crystal structure of the homologous bovine
soluble Rieske protein fragment (ISF), it seems
plaus-ible that the apo-protein may adopt its almost terminal
tertiary structure, as the cluster is bound only by a
minor subdomain on top of the b-sandwich fold [50]
Furthermore, a CD spectrum of the heterologously
expressed and refolded apo-ISF shows secondary
structure features similar to the native holo-protein
However, native PAGE indicates a partially mobile or
disordered structure for the refolded apo-ISF [48] In
addition, the Rieske protein carries a cystine bridge,
and it was shown that various disulfide-containing
pro-teins may be exported by the Tat pathway only under
conditions in which a mutant strain provides an
oxid-izing cytosolic environment [17] However, it is
reason-able to assume that disulfide bonds are formed in the
periplasm, catalysed by homologues of the DsbA⁄ B
machinery Therefore, we argue that the cystine bridge
is not essential for an export-competent structure of
the ISP In summary, it seems that the apo-ISP can
adopt its tertiary structure to a large extent and might
well be accepted by the Tat translocon; however,
disor-dered elements (the cluster binding subdomain) may
hamper this process
Genetic inactivation of Tat machinery components
in P denitrificans will be an interesting goal for future experiments Respiration is obligatory for this bac-terium [29]; however, a Tat-inactivated strain should
by viable under oxic conditions, given the bioenergetic flexibility of P denitrificans The expected defect of the cytochrome bc1 complex can be bypassed by the
ba3 quinol oxidase If such a mutant can be obtained,
it will certainly provide valuable information about the assembly of redox proteins in this important model system for the study of respiratory chains Another interesting outlook is the identification of a putative cytosolic binding partner of the ISP, e.g
by using chemical cross-linking approaches and MS, giving interesting insights into the assembly of Rieske proteins
Experimental procedures
Bioinformatic tools
Protein sequences were obtained from Swiss-Prot pro-tein database (http://www.expasy.org/sprot); (a) bacterial Rieske proteins: R rubrum P23136, R capsulatus P08500,
R sphaeroides Q02762, R viridis P81380, B japonicum P51130, P denitrificans P05417; (b) [NiFe] hydrogenase small subunits: A chroococcum P18190, A hydrogenophilus P33375, B japonicum P12635, R capsulatus P15283; (c) MauM ferredoxins: M extorquens Q49130, M flagellatum Q50423, M methylotrophus Q50235, P denitrificans Q51659; (d) NapA periplasmic nitrate reductases: A eutro-phus P39185, D desulfuricans P81186, R sphaeroides Q53176, P pantotrophus Q56350; (e) NosZ nitrous oxide reductases: A eutrophus Q59105, P aeruginosa Q01710,
P denitrificans Q51705, P stutzeri P19573, R meliloti Q59746; (f) TorA trimethylamine-N-oxide reductases:
E coli P33225, R capsulatus Q52675, R sphaeroides Q57366 Multiple sequence alignments were performed using clustal x v 1.81 [27] For secondary structure prediction based on multiple alignments, the web server JPRED [51] (http://www.compbio.dundee.ac.uk) was used Kyte–Doolittle hydropathy plots [25] were generated using an online tool from the ExPASy molecular bio-logy server (http://www.expasy.org/tools/protscale.html); the window size was set to 19 residues for comparison
of ISP sequences and the comparison group of Tat-translocated proteins Differences in hydropathy were statistically assessed with a two-tailed Mann–Whitney U-test (http://eatworms.swmed.edu/leon/stats/utest.html)
To estimate the hydropathy of TM helices, a limited collection of P denitrificans TM proteins was examined: cytochrome c552, cytochrome c1, cytochrome b and cyto-chrome c oxidase SU II A set of 20 predicted Sec-exported proteins was obtained from the SPDb server