When the second periplasmic loop was extended by an epitope tag to 27 amino acid residues, the membrane inser-tion of this loop of KdpD depended on SecE and YidC.. However, when the two
Trang 1The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC
Sandra J Facey and Andreas Kuhn
Institute of Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany
KdpD is a sensor kinase protein in the inner membrane of
Escherichia coli containing four transmembrane regions
The periplasmic loops connecting the transmembrane
regions are intriguingly short and protease mapping allowed
us to only follow the translocation of the second periplasmic
loop The results show that neither the Sec translocase nor
the YidC protein are required for membrane insertion of the
second loop of KdpD To study the translocation of the first
periplasmic loop a short HAepitope tag was genetically
introduced into this region The results show that also the
first loop was translocated independently of YidC and the
Sec translocase We conclude that KdpD resembles a new
class of membrane proteins that insert into the membrane
without enzymatic assistance by the known translocases
When the second periplasmic loop was extended by an
epitope tag to 27 amino acid residues, the membrane
inser-tion of this loop of KdpD depended on SecE and YidC To test whether the two periplasmic regions are translocated independently of each other, the KdpD protein was split between helix 2 and 3 into two approximately equal-sized fragments Both constructed fragments, which contained KdpD-N (residues 1–448 of KdpD) and the KdpD-C (residues 444–894 of KdpD), readily inserted into the membrane Similar to the epitope-tagged KdpD protein, only KdpD-C depended on the presence of the Sec translo-case and YidC This confirms that the four transmembrane helices of KdpD are inserted pairwise, each translocation event involving two transmembrane helices and a periplas-mic loop
Keywords: Escherichia coli; membrane protein; protein translocation; epitope tag
The inner membrane protein KdpD of Escherichia coli is
involved in osmoregulation It comprises of 894 amino acid
residues organized as two hydrophilic domains that are
separated by four closely spaced transmembrane regions [1]
KdpD is functionally related to other sensor kinases like
PhoR and EnvZ and shows a moderate sequence homology
in parts of the C-terminal domain with other sensor kinases
In the membrane, the KdpD protein forms a homodimer,
which has been proposed to be required for the kinase
function [2] The transmembrane regions are necessary for
signal perception because mutants in the transmembrane
regions have been found that are defective in the osmotic
response [3] To understand how the transmembrane helices
or the periplasmic loops sense an osmotic signal a precise
knowledge of the topology and membrane insertion of these
hydrophobic regions is crucial Intriguingly, the two
peri-plasmic loops separating the transmembrane regions
com-prise of only four and 10 amino acid residues, respectively
Multi spanning membrane proteins contain several
hydrophobic regions linked by hydrophilic loops of various
lengths ranging from a few amino acids to several hundred
residues, e.g in SecD [4] Long periplasmic loops are translocated by the ATP-driven Sec translocase, whereas small loops may be translocated by a synergistic mechanism without the Sec translocase as has been observed for the double-spanning M13 procoat protein [5,6] Based on results from a functional approach [7], a Sec-independent insertion has also been suggested for melibiose permease, which has six short periplasmic loops Gafvelin and von Heijne [8] have shown, through studying a tandem construction of leader peptidase that spans the membrane four times, that short periplasmic loops of about 25 residues were translocated independently of SecA, whereas long loops of 250 residues required the SecA-driven translocase However, De Gier et al [9] found by using the tightly controlled SecE mutant strain, that the SecYE translocase may be involved in the translocation of a 25 residue periplasmic loop The authors suggested that the hydro-phobicity of the transmembrane region determines the requirement of the Sec translocase
Proteins that are destined to be translocated across or inserted into the bacterial inner membrane are targeted to the translocation sites by multiple mechanisms In E coli, secretory proteins are targeted to the inner membrane by means of the chaperone SecB, which directs the newly synthesized protein to the SecAsubunit of the translocase complex of the Sec pathway, and whose membrane-integrated components are SecY, E, and G [10] In contrast, polytopic membrane proteins are targeted to the membrane
by an essential ribonucleoprotein complex that is closely related to the eukaryotic signal recognition particle (SRP)
E colicontains Ffh (P48), which together with 4.5S RNA,
Correspondence to A Kuhn, Institute of Microbiology and
Molecular Biology, University of Hohenheim, 70599 Stuttgart
Germany Fax: + 49 711 4592238, Tel.: + 49 711 4592222,
E-mail: andikuhn@uni-hohenheim.de
Abbreviations: HA, haemagglutinin; SRP, signal recognition particle;
IPTG, isopropyl 1-thio-b- D -galactoside; CCCP, carbonyl cyanide
p-chlorophenylhydrazone; pmf, proton motive force.
(Received 11 December 2002, accepted 20 February 2003)
Trang 2represents the bacterial homologue of the SRP [10].
Membrane translocation is then catalysed by SecY and
SecE; SecAand SecG are not required for most membrane
proteins [11]
Anew bacterial membrane protein insertion pathway was
recently discovered involving YidC, a protein homologous
to the mitochondrial Oxa-1p YidC was found to be
required for the insertion of Sec-independent membrane
proteins and is also involved in the membrane integration of
Sec-dependent proteins, whereas exported proteins, such as
OmpA, were not affected (reviewed in [12]) In the absence
of YidC, Sec-independent proteins accumulated at the
cytoplasmic side of the membrane, whereas Sec-dependent
membrane proteins were jammed in the Sec translocase
[13,14]
To understand the translocation process of
multispan-ning membrane proteins, we have investigated the
mechan-ism of how the sensor kinase protein KdpD inserts into the
membrane We found that KdpD inserts into the membrane
independently of the Sec translocase and YidC However,
when the two small periplasmic regions of the protein were
extended by short epitopes, we found that the translocation
of the first periplasmic region was still independent of the
Sec translocase and YidC, but the second extended
periplasmic region required the Sec translocase and YidC
Unexpectedly, the introduction of the epitope tag into the
second periplasmic region was the main cause for the
requirement for YidC
Materials and methods
Plasmid constructions
K Jung and K Altendorf (Universita¨t Osnabru¨ck,
Germany) kindly provided the plasmids, pPV5 and pBD
carrying the kdpD gene in pKK233-3 and pBAD18,
respectively [15,16] The strategy to generate the two
truncated halves of the protein was to cut KdpD in
approximately the middle between helix 2 and 3 By means
of site-directed mutagenesis, a stop codon (TAG) and an
NdeI restriction site was introduced between helix 2 and 3
The constructed fragments containing KdpD-N (i.e coding
the amino acid residues 1–448 of KdpD) and KdpD-C (i.e
coding the amino acid residues 444–894 of KdpD) were
cloned into the expression vector pT7-7
The epitope tags within the fragments were constructed
by first introducing a MunI restriction site between the first
and second and between the third and fourth helices by
site-directed mutagenesis The epitope tags were introduced into
the opened MunI sites of the respective plasmids by ligating
two short complementary oligonucleotides with AATT
overhangs These complementary oligonucleotides code
either for a haemagglutinin (HA)- or a T7-epitope tag with
a spacer of four amino acid residues Each of the tagged
constructs was sequenced to confirm the correct in-frame
fusion of the epitope cassettes
Strains, plasmids, and growth conditions
Cloning and mutagenesis experiments were performed with
E coliXL1-Blue recA1 thi supE44 endA1 hsdR17 gyrA96
relA1 lacF¢ (proAB+lacIqlacZDM15 Tn10) (Stratagene)
The pT7-7 expression vector with the kdpD gene was transferred into the E coli BL21(DE3)pLysS strain which expresses the T7 RNApolymerase under the inducible lacUV5 promoter [17]
The SecE-depletion strain CM124 [18] was cultured in M9 minimal medium supplemented with 0.4% glucose and 0.2% L-arabinose To deplete cells for SecE, overnight cultures were washed once with M9 medium and back-diluted 1 : 20 into fresh M9 medium in the absence of
L-arabinose Depletion of SecE was checked by monitoring the accumulation of the precursor to the outer membrane protein A(proOmpA)
The YidC-depletion strain JS7131 [13] was cultured in Luria–Bertani medium supplemented with 0.2% arabinose
To deplete cells for YidC, overnight cultures were grown in 0.2% arabinose and then washed twice with LB to remove cells of arabinose and back-diluted 1 : 50 into fresh Luria– Bertani medium with 0.2% glucose Depletion of YidC was checked by immunoprecipitating the labelled cells with antibodies to YidC
Media preparation and bacterial manipulations were performed according to standard methods [19] Where appropriate, ampicillin (100 lgÆmL)1, final concentration), kanamycin (50 lgÆmL)1, final concentration) and chloram-phenicol (25 lgÆmL)1, final concentration) were added to the medium
Wild-type KdpD, KdpD containing the HA- and T7-epitope tags, KdpD-N containing the N-terminal fragment with the HA-epitope and KdpD-C containing the C-terminal fragment with the T7-epitope were expressed
byL-arabinose induction from the pBAD18 vector [20] in strain MC1061 and by isopropyl thio-b-D-galactoside (IPTG) induction from the vectors pT7-7, pMS119 [21] and pDHB5700 [9] in strains BL21(DE3)pLysS, JS7131 and CM124, respectively
Antibodies The T7-tag monoclonal antibody recognizing the 11 amino acid T7 peptide (MASMTGGQQMG) was purchased from Novagen The anti-HArecognizes the HApeptide sequence (YPYDVPDYA) derived from the human influenza HA protein [22] The anti-HAmonoclonal antibody was purchased from Boehringer Polyclonal antibody against KdpD was a gift from K Jung and K Altendorf (Universita¨t Osnabru¨ck, Germany)
Protease mapping assay For all experiments, cells were grown to midlogarithmic phase Cells harboring the plasmid-encoded proteins were induced for 10 min either with IPTG (1 mM, final concen-tration) or for 1 h withL-arabinose (0.2%, final concentra-tion) Unless otherwise stated, cells were labelled with [35S]methionine for 5 min and chased with excessL -methio-nine for 5 min For spheroplasting, cells were centrifuged at
12 000 g and resuspended in 500 lL of ice-cold spheroplast buffer (40% w/v sucrose, 33 mMTris/HCl, pH 8.0) Lyso-zyme (5 lgÆmL)1, final concentration) and EDTA(1 mM, final concentration) were added for 15 min Aliquots of the spheroplast suspension were incubated on ice for 1 h either in the presence or absence of proteinase K (0.5 mgÆmL)1final
Trang 3concentration) Alysis control was included by adding 2.5%
Triton X-100 and proteinase K for 1 h After addition of
phenylmethanesulfonyl fluoride (0.33 mgÆmL)1, final
con-centration), samples were precipitated with trichloroacetic
acid (20%, final concentration), resuspended in 10 mMTris/
2% SDS, pH 8.0 and immunoprecipitated with antibodies
against HA, T7, KdpD, OmpA (a periplasmic control), or
GroE (a cytoplasmic control, results not shown) Samples
were analysed by SDS/PAGE and phosphorimaging
For the azide and carbonyl cyanide
p-chlorophenyl-hydrazone (CCCP) studies, the cells (0.5 mL cultures) were
pretreated by the addition of 10 lL of sodium azide
(100 mM) for 5 min or by the addition of 2.5 lL of CCCP
(10 mM) for 45 s, prior to labelling of the cells
Results
Membrane insertion of the KdpD protein
The membrane insertion of the KdpD protein is difficult to
analyse because the translocated periplasmic regions are
comprised of only four and 10 amino acid residues,
respectively We observed that proteinase K did not cleave
the protein in the first periplasmic loop, probably because
this loop is too short and does not extend far enough away
from the membrane surface to be accessible to the protease
Cleavage in the second periplasmic loop occurred partially
and led to a protease protected fragment of 47 kDa that
was recognized by the KdpD antibody that detects the
C-terminal cytoplasmic domain The generation of the
protease protected fragment allowed the investigation of
how the second (10 amino acid residues long) periplasmic
region of the wild-type KdpD is translocated
First, the involvement of SecAwas investigated using
sodium azide (Fig 1A) Sodium azide has been shown to
inhibit SecAactivity at 2 mMconcentration [23] To address
the role of SecAin KdpD membrane insertion, bacteria
were treated with 2 mM sodium azide for 5 min prior to
[35S]methionine addition After a pulse of 5 min, a fraction
of the radioactively labelled KdpD protein was accessible to
proteinase K added to the outside of the cells either in the
absence or presence of sodium azide (Fig 1A, lower panel)
Translocation of the second periplasmic loop of KdpD was
followed by the generation of the C-terminal 47 kDa
proteolytic fragment The results show that its formation
was not affected when the function of SecAwas perturbed
by azide (compare lanes 2 and 5) Following lysis of the cells
with detergent, we confirmed that the smaller fragment was
readily digested (lanes 3 and 6) As expected, proOmpA was
rapidly converted to OmpAin the absence of azide (upper
panel, lane 1) In the presence of azide, the Sec-dependent
proOmpAaccumulated in the cytoplasm of the cells and
was not digested by the protease (lanes 4 and 5)
To test the role of integral translocase components, the
involvement of SecE in KdpD membrane insertion was
investigated This was performed by using the strain
CM124, in which SecE can be depleted efficiently In this
strain, the secE gene expression is under the control of the
arabinose-inducible araBAD promoter [24] In the presence
of the repressor glucose and absence of arabinose, SecE is
not expressed CM124 cells were grown in the presence of
glucose or arabinose, respectively, and analysed for KdpD
membrane insertion When SecE was depleted, KdpD was still inserted because the proteolytic fragment was detectable
in equal amounts (Fig 1B, lower panel; compare lanes 2 and 5) As a control, the translocation of proOmpA was monitored (upper panel) As expected, proOmpA translo-cation was blocked under SecE-depleted conditions and not digested by the protease
The dependence of KdpD insertion on the proton motive force (pmf) was studied after treatment of the cells with CCCP, a protonophore that dissipates the pmf [25] The pmf was collapsed by adding 50 lM CCCP, 45 s before labelling the cells with [35S]methionine CCCP reduced the efficiency of the translocation of the second periplasmic loop of KdpD as indicated by the reduced appearance of the C-terminal fragment (Fig 1C, lower panel; compare lanes 2 and 5) Immunoprecipitation with OmpAantiserum showed the accumulation of the nontranslocated precursor (proOmpA), which was not digested by proteinase K (Fig 1C, upper panel)
The role of YidC in the membrane insertion of KdpD was examined in the depletion strain JS7131, where YidC expression is under the control of an araBAD promoter and operator [13] YidC expression was induced with arabinose and tightly repressed in the presence of glucose To deplete YidC, the cells were grown for 3 h with glucose and then analysed for KdpD insertion (Fig 1D, lower panel) Under both conditions, KdpD inserted into the membrane as judged by the appearance of the C-terminal fragment (lanes
2 and 5) As a control, the accumulation of M13 procoat protein was analysed in a parallel culture (Fig 1D, upper panel) The results show that under YidC-depleted condi-tions procoat accumulated and was not digested by the protease Taken together, these results suggest that the second periplasmic loop of the wild-type KdpD protein is inserted into the membrane in the absence of SecA, SecE and YidC
Short epitopes introduced into the periplasmic regions allow the analysis of insertion events
To analyse the translocation of the two periplasmic regions
of KdpD in detail, short epitope tags were introduced into these regions (Fig 2) Oligonucleotide-directed insertion was used to introduce a 15 residue HA-tag derived from the human influenza haemagglutinin protein between helix 1 and 2 and a 17 residue T7-tag of the T7 major capsid protein between helix 3 and 4 Aspecific monoclonal antibody (anti-HAor anti-T7) was then used to monitor the location of the epitope-tagged region with respect to the KdpD protein in the membrane The KdpD protein with the epitope tags was readily digested by proteinase K in both periplasmic regions (Fig 3A) The periplasmic location of the epitope-tagged regions is consistent with the proposed membrane topology
of KdpD [1] and shows that now both regions are well exposed away from the membrane surface and easily accessible by the protease
To address the role of SecAin the membrane assembly of KdpD containing the HA- and the T7-epitopes in the respective loops, bacteria were treated with 2 mM sodium azide for 5 min prior to [35S]methionine addition Figure 3A (middle and lower panel) shows that both periplasmic loops
of KdpD are translocated in the absence (lane 2) and in the
Trang 4presence (lane 5) of sodium azide, under conditions in which
proOmpAtranslocation is reduced (Fig 3A, upper panel)
This suggests that SecAis not necessary for membrane
insertion of KdpD
The requirement of the Sec translocase was tested in the
CM124 strain where SecE is depleted when the cells are
grown in the absence of arabinose When the cells
expres-sing KdpD with the HA- and the T7-epitopes were grown in
the presence of glucose to deplete SecE (Fig 3B, middle and
lower panel), the membrane translocation of only the first
periplasmic loop of KdpD was efficient (middle panel,
compare lanes 2 and 5) The translocation of the second
periplasmic loop of KdpD was only about 70% efficient
indicating a dependence on SecE (Fig 3B, lower panel) In
the same cells, proOmpAexport was totally blocked by the
depletion of SecE (Fig 3B, upper panel) This differs from
the results obtained with the wild-type KdpD protein, where
the translocation of the second periplasmic loop without the
epitope tag was not affected by SecE depletion (Fig 1B)
To assess the effect of the pmf on the membrane insertion
of KdpD containing the epitope tags, the protonophor CCCP (50 lM) was added 45 s prior to pulse-labelling of the cells Figure 4A(middle and lower panels) shows the
Fig 1 The translocation of the second periplasmic loop of KdpD is
independent of SecA, SecE and YidC, but is sensitive to the membrane
potential (A) Protease mapping of KdpD in the absence (–) and
presence (+) of sodium azide to block SecAfunction E coli strain
MC1061 expressing the wild-type KdpD was grown at 37 C to
mid-log phase, induced for 1 h with 0.2% arabinose and labelled with
[35S]methionine for 5 min The cells were converted to spheroplasts
and incubated with (lanes 2 and 5) or without proteinase K (lanes 1
and 4) at a final concentration of 0.5 mgÆmL)1on ice for 1 h Alysis
control was included by adding proteinase K (0.5 mgÆmL)1, final
concentration) and 2.5% Triton X-100 (lanes 3 and 6) All samples
were precipitated with 20% trichloroacetic acid, immunoprecipitated
with antiserum to OmpA(upper panel) and KdpD (lower panel) and
analysed by SDS/PAGE and visualized by phosphorimaging The
positions of the molecular weight standards (SeeBlueTMPre-Stained
Standard, from Invitrogen) are marked on the right (B) Strain CM124
expressing KdpD was grown in M9 minimal medium containing
arabinose (lanes 1–3) For depletion of SecE (lanes 4–6), cells were
grown in the absence of arabinose for 8 h The cells were then induced
with 1 m M IPTG for 10 min Cells were pulse-labelled for 5 min and
chased with 500 lgÆmL)1 cold L -methionine for 5 min and
subse-quently analysed as described as above As a control, proOmpA
processing was monitored in parallel to verify SecE depletion (C)
Protease mapping of KdpD in the absence (–) and presence (+) of the
protonophore CCCP to dissipate the pmf CCCP was added 45 s prior
to labelling at a final concentration of 50 l M E coli MC1061 bearing
pBAD18 encoding wild-type KdpD was induced with arabinose for
1 h, labelled with [ 35 S]methionine for 5 min and chased with
500 lgÆmL)1cold L -methionine for 5 min as described above
Clea-vage of proOmpAwas monitored as a control (upper panel) (D) To
test the requirement of YidC, the YidC depletion strain JS7131 was
induced with arabinose or tightly repressed in the presence of glucose.
E coli strain JS7131 containing the cloned kdpD gene (pMS119kdpD)
was grown in LB with either 0.2% arabinose (YidC + ) or 0.2% glucose
(YidC – ) for 3 h One millimolar IPTG was added for 10 min to induce
expression and the cells were pulse-labelled for 1 min, then converted
to spheroplasts by lysozyme treatment and osmotic shock
Translo-cation of the YidC-dependent M13 coat protein was monitored in
parallel by proteinase K treatment of spheroplasts (upper panel).
Samples were immunoprecipitated with antiserum to M13 coat protein
(upper panel) and with antiserum to KdpD, respectively (lower panel).
Trang 5membrane translocation of the periplasmic loops of KdpD
in the absence and in the presence of CCCP These results
demonstrate that the pmf is required for efficient membrane
insertion of KdpD with the tags This is in agreement with
the wild-type KdpD, which is also sensitive to the pmf for
efficient membrane assembly (Fig 1C, lower panel)
We also investigated the involvement of YidC for the
translocation of KdpD with the two epitopes in the
YidC-depleted strain JS7131 Figure 4B (middle panel) shows that
in cells grown with glucose to deplete YidC, the first
periplasmic loop was normally translocated and did not
differ from the cells grown with arabinose (compare lanes 2
and 5) The translocation of the second periplasmic loop
(Fig 4B, lower panel), however, was affected in the cells
with depleted YidC This indicates that the two periplasmic
loops of KdpD with the epitope tags are translocated
differently Whereas the first loop translocates in the
absence of SecA, SecYE and YidC, but depends on the
pmf, the translocation of the second loop is supported by
SecYE and YidC
Membrane insertion of split osmosensor fragments
The kdpD gene encoding the HA- and the T7-epitopes was
split into 2 approximately equal-sized fragments between
helix 2 and 3 The constructed fragments containing
KdpD-N (i.e coding the amino acid residues 1–448 of KdpD) and
KdpD-C (i.e coding the amino acid residues 444–894 of
KdpD) were subcloned into pT7-7 The KdpD fragments were stably expressed as truncated N- or C-terminal halves, each with double-spanning membrane helices
As described above, we used the protease accessibility assay to analyse the insertion of the KdpD truncated halves into the membrane Both truncated halves, termed KdpD-N and KdpD-C, were readily inserted into the inner mem-brane and the epitopes were digested by the externally added protease Intriguingly, a stable dimeric form was observed only for KdpD-N (Fig 5A) The membrane
Fig 3 The involvement of SecA (A) and SecE (B) in the translocation of the individual membrane loops (A) The kdpD gene containing the epitope tags was expressed in strain MC1061 in the presence (lanes 1–3) or absence (lanes 4–6) of sodium azide Cells were pulse-labelled with [ 35 S]methionine for 5 min and then converted to spheroplasts as described in the legend to Fig 1 The epitope-tagged KdpD protein was immunoprecipitated with antiserum to HA(for the epitope in the first periplasmic loop; middle panel) and to T7 major capsid protein (for the epitope in the second periplasmic loop; lower panel), respect-ively, and then analysed by SDS/PA GE and visualized by phos-phorimaging OmpAaccumulated in its precursor form (proOmpA)
in the azide treated cells (upper panel, lanes 4–5) (B) CM124 cells expressing the epitope-tagged KdpD were pulse-labelled with [ 35 S]methionine for 5 min and chased for 5 min either in the presence
of arabinose to induce expression of SecE (lanes 1–3) or in the absence
of arabinose to deplete SecE (lanes 4–6) Translocation of the Sec-dependent protein OmpAwas monitored in parallel after a 1-min pulse-labelling (upper panel).
Fig 2 Membrane topology of KdpD (A) and introduction of epitopes to
extend the short periplasmic regions of KdpD (B) (A)
Oligonucleotide-directed mutagenesis was used to integrate a HA-epitope derived from
the human influenza haemagglutinin protein into the first periplasmic
loop of KdpD and a T7-epitope of the T7 major capsid protein into the
second periplasmic loop of KdpD (B) lists the amino acid sequences of
each of the two extra-membrane loops before and after the insertion of
the epitopes Insertion of the epitopes (underlined) has the following
consequences for length (number of amino acid residues) and net
charge of the loops (without/with tag); Helix 1/2: (4/19) ( )1/)3); Helix
3/4: (10/27) (0/0).
Trang 6insertion of the N- and C-terminal halves was then studied
in CM124 cells where SecE was depleted (Fig 5A,B) The
cells were induced, labelled with [35S]methionine for 5 min,
chased for 5 min, immediately converted to spheroplasts
and treated with proteinase K The samples were
immuno-precipitated with antibodies to the respective tags (anti-HA
or anti-T7) and analysed by SDS/PAGE, and the bands
were visualized on a phosphorimager The translocation of
KdpD-N was not affected by the depletion of SecE
(Fig 5A), whereas KdpD-C was clearly affected by the
SecE depletion (Fig 5B) In both experiments, the trans-location and cleavage of proOmpAwas efficiently blocked when SecE was depleted (upper panels) In agreement with the results obtained from studies with the four-spanning KdpD protein containing the epitope tags (Fig 3B), the first periplasmic loop was translocated across the membrane in a Sec-independent fashion, whereas the translocation of the second periplasmic loop with the tag indicated a dependence
on SecE for efficient insertion
Membrane potential is required for the insertion
of KdpD-N
To test whether the translocation of the periplasmic loops requires the pmf, the location of the loops was analysed in the presence of CCCP As shown in Fig 6A, CCCP completely blocked translocation of KdpD-N The protein was not accessible to the externally added proteinase K, indicating that it remains in the cytoplasm Intriguingly, the formation of the dimeric form was also blocked In contrast, the membrane insertion of KdpD-C was partially affected
by the addition of CCCP (Fig 6B), and most of the protein
Fig 4 The involvement of the electrochemical membrane potential (A)
and YidC (B) in the translocation of the individual membrane loops (A)
Proteinase K mapping of the epitope-tagged KdpD protein in the
absence (–) and presence (+) of CCCP E coli MC1061 cells bearing
the pBAD18-plasmid coding for the epitope-tagged KdpD protein
were labelled with [ 35 S]methionine for 5 min at 37 C and chased with
500 lgÆmL)1L -methionine for 5 min Cells were then converted to
spheroplasts and analysed as described in Fig 3 Dissipation of the
membrane potential was checked by monitoring the accumulation of
proOmpA (B) Proteinase K mapping of the epitope-tagged KdpD
protein in the YidC depletion strain, JS7131 Cells were grown in the
presence of arabinose (YidC + ) or in the presence of glucose (YidC – )
and pulse-labelled for 5 min The cells were then converted to
spheroplasts and treated with or without proteinase K for 1 h, and
analysed as described in Fig 3 OmpAprocessing was monitored in
parallel after a 1-min pulse-labelling (upper panel).
Fig 5 Effects of SecE depletion on the translocation of the split KdpD proteins CM124 cells expressing KdpD-N (A) or KdpD-C (B) were grown in M9 minimal medium either in the presence (SecE+) or absence of arabinose (SecE – ) Cells were pulse-labelled with [35S]methionine for 5 min and chased for 5 min with 500 lgÆmL)1
L -methionine and analysed as outlined in the legend to Fig 1 Samples were immunoprecipitated with antiserum to HA(for KdpD-N) and to T7 major capsid protein (for KdpD-C), respectively OmpAprocessing was monitored in parallel to check spheroplasting and SecE depletion (upper panels) The extra band observed in the lower part of Ais the dimer of KdpD-N.
Trang 7was accessible to proteinase K This demonstrates that the
pmf is required for the insertion of KdpD-N, but has only a
slight effect on KdpD-C
YidC is required for efficient insertion of KdpD-C,
but not for KdpD-N
We investigated the effect of YidC depletion on the
translocation of KdpD-N and KdpD-C in the strain
JS7131 When YidC was present (cells grown with
arabi-nose), both proteins were readily inserted into the membrane
and digested with proteinase K (Fig 7Aand B, lanes 1 and
2) In YidC-deficient cells (grown with glucose), KdpD-N
inserted normally into the membrane and was digested with
proteinase K (Fig 7A, compare lanes 2 and 5) Likewise,
dimer formation was also not affected Therefore,
translo-cation of KdpD-N is independent of YidC
In contrast, the translocation of KdpD-C with the
T7-epitope was affected in the cells grown with glucose,
indicating a dependence on YidC for efficient insertion
(Fig 7B) When YidC was not depleted (YidC+), KdpD-C
was efficiently inserted and digested with proteinase K
(lanes 1 and 2) Because the wild-type KdpD protein without the tags was inserted independently of YidC (Fig 1D), the introduction of an epitope tag might affect the membrane insertion
To test this, the membrane insertion of KdpD-C with (Fig 8A) and without the epitope tag (Fig 8B) was followed with the KdpD antibody which recognizes the C-terminal cytoplasmic domain of KdpD Therefore, if the periplasmic loop is cleaved by the protease, only a small shift of the molecular mass of the protein is expected because the antibody recognizes the remaining C-terminal domain In the presence of YidC, the shift of the molecular mass of KdpD-C with the tag was complete when protei-nase K was added externally (Fig 8A, lane 2) When the cells were depleted for YidC, the generation of the shift was inhibited showing that the periplasmic loop was not translocated (Fig 8A, compare lanes 2 and 5) In contrast, the untagged KdpD-C was only partially shifted (Fig 8B) This is because the short periplasmic region is not well exposed at the cell surface, in agreement with the observa-tions from the wild-type KdpD When YidC was depleted, membrane insertion of KdpD-C without the epitope tag appeared almost as efficient as that of the YidC-containing cells (Fig 8B, compare lanes 2 and 5) Taken together, these results suggest that the presence of the epitope tag is the reason why KdpD-C requires the assistance of YidC
Discussion
The present study was initiated to understand how multi-spanning membrane proteins with short periplasmic loops are inserted into the membrane bilayer Most studies on
Fig 6 The KdpD-N fragment (A) requires the electrochemical
mem-brane potential for memmem-brane insertion, whereas the KdpD-C fragment
(B) is only slightly affected MC1061 cells with plasmids expressing the
mutant proteins were analysed with (+) or without CCCP (–) as
described in the legend to Fig 1 Cells bearing plasmids encoding these
proteins were pulse-labelled with [ 35 S]methionine for 5 min and chased
for 5 min OmpAaccumulated in its precursor form (proOmpA) in
CCCP treated cells (upper panels, lanes 4–5).
Fig 7 YidC is required for efficient membrane insertion of KdpD-C (B) but not for KdpD-N (A) Plasmids encoding KdpD-N (A) or KdpD-C (B) were transformed into E coli JS7131 The cells were analysed in pulse-labelling experiments under YidC-depleted or YidC-expressing conditions as described in Fig 1 After subjecting the cells to a pro-tease accessibility assay, the proteins were immunoprecipitated with antiserum to HA(A), to T7 major capsid protein (B) and analysed by SDS/PAGE and phosphorimaging.
Trang 8multispanning proteins made so far have focussed on the
translocation of large domains [26–28] Short periplasmic
regions are difficult to analyse, since they hide as an
antigenic target and resist proteolytic assessment [29–31]
We used the four-spanning membrane protein KdpD as a
model system It contains two periplasmic loops of four and
10 amino acid residues The first periplasmic region of
KdpD proved resistant to proteinase K, whereas the second
periplasmic loop of the KdpD protein was partially
accessible to externally added protease and the digestion
resulted in a smaller C-terminal fragment We found that
only about 50% of the protein was digested by the protease
When the periplasmic region was extended by 17 amino acid
residues, more than 95% of the protein was accessible,
suggesting that the short periplasmic region in KdpD is
affected in its surface exposure, not in its membrane
translocation The analysis of the membrane insertion of
the wild-type KdpD showed that the translocation of the
second periplasmic loop is independent of SecA, SecE, and
YidC, and is only affected by the loss of the membrane
potential (Fig 1)
To analyse the translocation of the two periplasmic
regions of KdpD short epitopes were introduced into these
regions Antibodies specific for each epitope were used for
immunoprecipitation showing that the translocation of
both periplasmic loops can be analysed individually This
enabled the testing of whether the Sec translocase is
involved in the membrane insertion process Using the
strain CM124, where the SecE content can be extensively
depleted [9], we observed that the first periplasmic loop of
KdpD was translocated normally across the membrane
(Fig 3B) Because in the absence of SecE, SecY is rapidly
degraded [32], we conclude that the translocation of the first
loop is independent of SecYE Likewise, the inactivation of SecAby azide [23] did not affect the membrane insertion, suggesting that wild-type KdpD is inserted Sec-independ-ently This is different to most other known membrane proteins that require at least the integral components of the Sec translocase for membrane insertion Mannitol permease and SecY require SecYE for insertion, but are independent
of SecAand SecG [33], whereas leader peptidase and YidC require SecYEG and SecA[34–36] The different require-ments suggest that translocation components function as modules responsible for specific tasks For example, leader peptidase has a large C-terminal domain in the periplasm that requires SecAin addition to SecYEG [37] Similarly, large periplasmic loops extending 100 amino acid residues in M13 procoat mutants, need SecAand SecYE for translo-cation, whereas small loops do not stimulate the transloca-tion ATPase of SecA [6,38] The result obtained here that KdpD is independent of SecAis therefore consistent with previous findings
The results obtained for the KdpD protein showed that the use of short epitopes can provide valuable data for the analysis of how specific regions of a membrane protein are translocated across the membrane The analysis of the translocation requirements showed that the first periplasmic loop of KdpD with the epitope tag was independent of the Sec components, whereas the longer second periplasmic loop of KdpD required SecE and YidC for efficient translocation (Figs 3B and 4B) This indicates that the multispanning membrane protein actually translocates in pairs of transmembrane helices and that individual pairs may have different insertion requirements, depending on the connecting loops Interestingly, the two translocation events observed for KdpD with the epitope tags correspond to
Fig 8 YidC is required for efficient membrane
insertion of KdpD-C with the epitope tag (A)
but not for KdpD-C without the epitope tag (B).
JS7131 cells bearing the pMS119 plasmids
encoding either KdpD-C with the T7 epitope
tag (A) or KdpD-C without the tag (B) were
depleted of YidC as described in the legend
of Fig 1 After subjecting the cells to a
protease accessibility assay, the proteins were
immunoprecipitated with antiserum to KdpD
and analysed by SDS/PA GE and
phosphori-maging PK, proteinase K.
Trang 9those of the split double-spanning proteins (Fig 5) This
underlines that membrane proteins are inserted not in a
linear movement, but rather as individual domains
Experi-ments with leader peptidase had shown earlier that the
N-terminal tail and the large C-terminal domain are
separately translocated [39] The pairwise organization of
multispanning membrane proteins is also suggested from
single-molecule force spectroscopy where a molecular
tweezer was connected to the C-terminus of
bacteriorho-dopsin [40] When the protein was pulled out of the
membrane, two transmembrane regions were preferentially
released together
Unexpectedly, YidC is not important for the membrane
insertion of the KdpD wild-type protein (Fig 1D) Other
Sec-independent proteins, such as Pf3 coat and M13
procoat strongly depend on YidC [14,41] In contrast to
KdpD, the M13 procoat protein has a periplasmic region of
20 amino acid residues including five charged residues
Interestingly, different mutants with alterations in the loop
region of procoat have shown that the number of the
charged residues determines the extent of YidC dependency
Amutant that has no charged residue in the 20 amino acid
loop showed only a minor interference by YidC depletion
[14] This might explain why KdpD is independent of Sec
and YidC as the periplasmic loops are much shorter and the
translocation of these periplasmic regions should require
less energy An extension of the second loop of KdpD by 17
amino acid residues indeed resulted in the requirement of
the YidC protein, suggesting that YidC promotes the
translocation of larger periplasmic regions
Interestingly, the two periplasmic loops of KdpD that
were extended with short epitope tags differed also for their
need of a membrane potential Whereas KdpD-N is not
translocated in the absence of a potential, KdpD-C was only
marginally affected Potential-dependent translocation of
negatively charged regions has been extensively studied with
the M13 procoat protein The periplasmic loop of the
procoat protein has a net negative charge of)3 Procoat
mutants were studied where the charge of the periplasmic
loop has been changed [42] Only the negatively charged
regions show potential dependence and the more negatively
charged residues present in the loop region of procoat the
higher is the potential dependency The procoat mutant
with a net charge of)1 in the periplasmic loop was only
marginally affected In agreement with this, the KdpD-N
protein with the HA-tag has three aspartic acyl residues in
the periplasmic loop, which might contribute to the strong
dependency on the membrane potential
For the Sec-independent Pf3 coat protein it was shown
that a mutant with a longer hydrophobic region inserts
independent of YidC and of the electrochemical membrane
potential [43,44] It was proposed that the hydrophobic effect
of the transmembrane region might drive the insertion step
and that this process can occur without any other protein
Under limited hydrophobicity, the electrochemical
mem-brane potential and YidC become then essential factors
These findings can be applied to the insertion of the KpdD
protein If a protein can autonomously insert into the
membrane, the hydrophobic energy from the insertion of
the hydrophobic parts of the protein should compensate the
energy costs of the transfer of its hydrophilic part Taking the
hydrophobicity scale [43] to calculate the free energy that
the transmembrane regions of KdpD can contribute to the membrane insertion we get about DG>
¼)144 kJÆmol)1for the first two helices The transfer of the periplasmic loop between these helices to translocate costs DG>
¼
65 kJÆmol)1, which should allow an autonomous insertion However, when the HA-tag is included in the hydrophi-lic region the energy cost increases to about DG>
¼
200 kJÆmol)1 This would not allow membrane insertion and might explain the strong dependence of KdpD-N on the pmf The membrane insertion of the helices 3 and 4 con-tributes with only DG>
¼)63 kJÆmol)1 The second peri-plasmic loop of the wild-type costs DG>
¼ 105 kJÆmol)1, and with the added T7-epitope DG>
¼ 150 kJÆmol)1
is required to pass the membrane The hydrophobic contri-bution cannot compensate the energy costs of the transfer of the periplasmic loop This may explain why YidC and Sec play a role in the translocation of the C-terminal loop with the T7-epitope tag
Taken together, the data presented here show that KdpD inserts unassisted from the Sec translocase and YidC into the inner membrane of E coli This is most likely because KdpD has very short periplasmic regions that cost little energy to translocate suggesting that the membrane inser-tion occurs autonomously The unassisted inserinser-tion path-way may also be used by a large number of E coli membrane proteins with short periplasmic loops that have not yet been analysed for membrane insertion So far, the unassisted membrane insertion pathway is known from thylakoids [45,46], where a subset of membrane proteins show independence of SRP, the Sec components and Alb3, the plant homologue of YidC
Acknowledgements
We would like to thank Drs K Jung and K-H Altendorf for generously providing us with the initial plasmids (pPV5, pBD) and KdpD antiserum and Drs H-G Koch, M Mu¨ller, R Dalbey and
D Kiefer for valuable discussions This work was supported by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 495.
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