The 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 HA epitope 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;membraneprotein;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 SecA subunit 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. coli contains 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) Eur. J. Biochem. 270, 1724–1734 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03531.x represents the bacterial homologue of the SRP [10]. Membrane translocation is then catalysed by SecY and SecE; SecA and SecG are not required for most membrane proteins [11]. A new 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 ¨ tOsnabru ¨ 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. coli XL1-Blue recA1 thi supE44 endA1 hsdR17 gyrA96 relA1 lac F¢ (proAB + lacI q lacZDM15 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 RNA polymerase 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 by L -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-HA recognizes the HA peptide sequence (YPYDVPDYA) derived from the human influenza HA protein [22]. The anti-HA monoclonal 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 m M , final concen- tration) or for 1 h with L -arabinose (0.2%, final concentra- tion). Unless otherwise stated, cells were labelled with [ 35 S]methionine for 5 min and chased with excess L -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 m M Tris/HCl, pH 8.0). Lyso- zyme (5 lgÆmL )1 , final concentration) and EDTA (1 m M , 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 )1 final Ó FEBS 2003 KdpD membrane insertion (Eur. J. Biochem. 270) 1725 concentration). A lysis 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 m M Tris/ 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 m M ) for 5 min or by the addition of 2.5 lLofCCCP (10 m M ) 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 SecA was investigated using sodium azide (Fig. 1A). Sodium azide has been shown to inhibit SecA activity at 2 m M concentration [23]. To address the role of SecA in KdpD membrane insertion, bacteria weretreatedwith2m M sodium azide for 5 min prior to [ 35 S]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 SecA was 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 OmpA in the absence of azide (upper panel, lane 1). In the presence of azide, the Sec-dependent proOmpA accumulated 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 l M CCCP, 45 s before labelling the cells with [ 35 S]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 OmpA antiserum 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. A specific monoclonal antibody (anti- HA or 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 SecA in the membrane assembly of KdpD containing the HA- and the T7-epitopes in the respective loops, bacteria were treated with 2 m M sodium azide for 5 min prior to [ 35 S]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 1726 S. J. Facey and A. Kuhn (Eur. J. Biochem. 270) Ó FEBS 2003 presence (lane 5) of sodium azide, under conditions in which proOmpA translocation is reduced (Fig. 3A, upper panel). This suggests that SecA is 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, proOmpA export 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 l M ) 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 SecA function. E. coli strain MC1061 expressing the wild-type KdpD was grown at 37 °Ctomid- log phase, induced for 1 h with 0.2% arabinose and labelled with [ 35 S]methionine for 5 min. The cells were converted to spheroplasts and incubated with (lanes 2 and 5) or without proteinase K (lanes 1 and4)atafinalconcentrationof0.5mgÆmL )1 on ice for 1 h. A lysis 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 (SeeBlue TM Pre-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 )1 cold L -methionine for 5 min as described above. Clea- vage of proOmpA was 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). Ó FEBS 2003 KdpD membrane insertion (Eur. J. Biochem. 270) 1727 membrane 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,andthenanalysedbySDS/PAGEandvisualizedbyphos- phorimaging. OmpA accumulated 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 OmpA was 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). 1728 S. J. Facey and A. Kuhn (Eur. J. Biochem. 270) Ó FEBS 2003 insertion 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 [ 35 S]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 proOmpA was 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 )1 L -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. OmpA processing 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 [ 35 S]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. OmpA processing was monitored in parallel to check spheroplasting and SecE depletion (upper panels). The extra band observed in the lower part of A is the dimer of KdpD-N. Ó FEBS 2003 KdpD membrane insertion (Eur. J. Biochem. 270) 1729 was 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. 7A and 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 membrane 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. OmpA accumulated 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. 1730 S. J. Facey and A. Kuhn (Eur. J. Biochem. 270) Ó FEBS 2003 multispanning 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 SecA by 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 SecA and 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 SecA in addition to SecYEG [37]. Similarly, large periplasmic loops extending 100 amino acid residues in M13 procoat mutants, need SecA and 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 SecA is 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 andanalysedbySDS/PAGEandphosphori- maging. PK, proteinase K. Ó FEBS 2003 KdpD membrane insertion (Eur. J. Biochem. 270) 1731 those 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. A mutant 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 )1 for 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.Thisismostlikelybecause KdpD has very short periplasmic regions that cost little energy to translocate suggesting that the membrane inser- tion occurs autonomously. The unassisted insertion 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. 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Albino3 J Biol Chem 276, 36200–36206 46 Woolhead, C.A., Thompson, S.J., Moore, M., Tissier, C., Mant, A., Rodger, A., Henry, R & Robinson, C (2001) Distinct Albino3-dependent and – independent pathways for thylakoid membrane protein insertion J Biol Chem 276, 40841– 40846 . The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC Sandra J. Facey and Andreas Kuhn Institute. 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.