• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Jun 1, 2010; 107(22): 10044–10049.
Published online May 17, 2010. doi:  10.1073/pnas.0914680107
PMCID: PMC2890433
Biochemistry

The action of cardiolipin on the bacterial translocon

Abstract

Cardiolipin is an ever-present component of the energy-conserving inner membranes of bacteria and mitochondria. Its modulation of the structure and dynamism of the bilayer impacts on the activity of their resident proteins, as a number of studies have shown. Here we analyze the consequences cardiolipin has on the conformation, activity, and localization of the protein translocation machinery. Cardiolipin tightly associates with the SecYEG protein channel complex, whereupon it stabilizes the dimer, creates a high-affinity binding surface for the SecA ATPase, and stimulates ATP hydrolysis. In addition to the effects on the structure and function, the subcellular distribution of the complex is modified by the cardiolipin content of the membrane. Together, the results provide rare and comprehensive insights into the action of a phospholipid on an essential transport complex, which appears to be relevant to a broad range of energy-dependent reactions occurring at membranes.

The implicit influence of the constitution and physical characteristics of the bilayer on the structure and assembly of membrane proteins is exemplified by a number of membrane-associated reactions with specific dependencies on lipids. In keeping with this, the bacterial protein translocation reaction, driven by the ATPase SecA through the conserved SecYEG complex, has a known requirement for acidic phospholipids (15).

The polar lipids of the Escherichia coli inner membrane consist of approximately 67% phosphatidylethanolamine (PE), 23% phosphatidylglycerol (PG), and 10% cardiolipin (CL) (6). The influence of the latter acidic pair on the SecA structure and activity has been well documented: SecA dimers form monomers upon contact with acidic phospholipids (5, 7, 8), as they do when bound to SecYEG (9). The association and activation of SecA at the cytosolic surface of the membrane requires constituent PG (1, 3). Finally, the allosteric inhibition of SecA by Mg2+, which prevents futile hydrolysis in the cytosol, is specifically relieved by CL (10).

Here we investigate the effects of lipids on the structure, activity, and localization of the SecYEG complex. The membrane bound and active version of SecYEG is dimeric (1114), but only one copy is necessary to create the channel (15). We show that CL, and to a lesser degree PG, promotes the formation of SecYEG dimers and a high-affinity binding surface for SecA. Once the complex is formed, the lipid also serves to stimulate high-level turnover of ATP. In the cell envelope, the propensity of SecYEG to localize into spiral-like structures is dependent on CL.

Results

A High-Affinity Association of CL with SecYEG Confers Its Ability to Activate SecA and Stimulates Preprotein Translocation.

The ATPase activity of SecA is stimulated by low concentrations of CL (10). To determine if the interaction is brokered through SecYEG, rather than from CL free in the membrane, we analyzed the lipid content of the purified complex and the consequences on its activity. SecYEG extracted from [32P]-exposed E. coli was analyzed by TLC and found to be significantly enriched for CL (Fig. 1A). Subsequent purification by gel filtration in the standard conditions (0.1% C12E9) retain PE and CL bound to the complex; with more stringent detergent regimes (0.5% C12E9) only CL remains, which is presumably very tightly bound (Fig. 1B, lanes 1 and 2).

Fig. 1.
Cardiolipin is tightly bound to SecYEG and is required for ATPase-translocation coupling. (A) An aliquot of [32P]-labeled membrane (~150,000 cpm) enriched for the SecYEG complex was solubilized with DDM and incubated with Ni2+-chelated ...

The lipid-depleted SecYEG preparation was then resupplemented with total E. coli polar lipids (PL) or just CL and repurified. The lipids bound to SecYEG were enriched, specifically by CL, which is present at only 10% in the PL mixture (Fig. 1 B and C). Moreover, the quantity of CL bound to SecYEG was proportional to its ability to stimulate the ATPase activity of SecA (Fig. 1C). Because the protein samples were isolated from CL micelles by gel filtration (Fig. S1), the stimulation of SecA must have been by CL associated with SecYEG.

CL is also a requirement for energy coupling and efficient protein translocation (Fig. 1 D and E). Proteoliposomes reconstituted with synthetic phospholipids to match the proportions found in E. coli support protein translocation of proOmpA and the associated ATPase activity, albeit to a lesser degree than the natural mixture. Removal of CL from the mixture uncouples ATPase activity from translocation; ATP turnover was unaffected whereas the ability to transport preprotein was severely impaired. This observation was not a result of variation in the reconstitution efficiency of SecYEG in the presence and absence of CL (Fig. S2).

CL Promotes the Association of SecA and SecYEG and Primes Them for Translocation.

The apparent binding parameters of SecYEG for SecA were derived from steady-state ATPase measurements in the presence of detergent-solubilized SecYEG with or without an excess of the different lipids (Fig. 2A). CL dramatically increases the affinity of SecYEG for SecA (35-fold) while increasing the ATP turnover (6.5-fold) (Table 1); the effect of PG was smaller (respectively, 19 and 2.3 times). This titration was repeated with double the concentration of PG (Fig. 2A), the equivalent to CL (composed of two glycerol-linked PGs), and the corresponding stimulation could not be achieved. Therefore, the acidic phospholipid effect is best served when the two PG molecules are covalently attached. In contrast, PE had no effect on the ATPase activation, whereas PL caused a small stimulation commensurate with its proportion of CL.

Fig. 2.
Cardiolipin confers tight SecYEG binding and high ATPase activity of SecA. (A) The ATPase activity of SecA was measured in detergent solution (0.03% C12E9) with increasing concentrations of SecYEG in the absence (Circles, Black Solid Line) or presence ...
Table 1.
Fitted SecA-SecYEG binding parameters in the absence or presence of various lipids from ATPase and fluorescence binding measurements (Figs. 2 and and55)

Next, the ATPase activity of SecA was measured with increasing amounts of CL, with or without saturating SecYEG (Fig. 2B). As expected, the magnitude of the stimulation was much higher in its presence and the apparent affinity of CL for the SecA-SecYEG complex was high (Kd[CL] = 9 μM from the fitting). The affinity for CL on SecA alone was much weaker; therefore, the apparent affinity could not be determined. Furthermore, the steady-state parameters KM and kcat (see * in Fig. 2B and Table 2) show that SecYEG and CL act synergistically on SecA, reducing ATP affinity (KM[ATP] from 0.32 to 3.9 μM) and increasing ATP turnover (kcat from 0.56 to 6.9 min-1). Changes of this nature prime SecYEG-SecA for translocation because they correspond to a shift of the enzyme toward the fully energy-transducing state, achieved when bound to the membrane and during preprotein transport (KM[ATP] = 46 μM and kcat = 460 min-1) (16).

Table 2.
Fitted kinetic parameters for ATPase activity obtained in the presence of the indicated molecules

Cardiolipin Stabilizes SecYEG Dimers.

To test whether or not the CL acts by modulating the oligomeric state of SecYEG, the complex was incubated with different phospholipids and analyzed by Blue Native PAGE (BN-PAGE) (Fig. 3A). Reducing the amount of detergent (from left to right) resulted in the formation of SecYEG dimers; CL was clearly the best lipid at promoting or stabilizing this (middle). Further reduction of the detergent induced aggregation (right, top part).

Fig. 3.
Cardiolipin facilitates dimerization of the SecYEG complex. (A) The [125I]-SecYEG complex (~30,000 cpm) was incubated on ice in TSG buffer containing 0.8% DDM and the indicated lipids. The molar ratio detergent/lipids (RDDM/Lip) was decreased ...

The dimer-promoting effect of CL was further assessed by using a nonspecific photo-cross-linking method (17), and various cross-linked products were obtained (Fig. 3B). The dimerization of the SecYEG complex, reflected by cross-links between two adjacent SecYs, was stimulated by lipids, particularly CL. The dimer stabilized by CL also forms a cross-link between two SecE subunits (Fig. 3B, lane CL*), consistent with the “back-to-back” arrangement of SecYEG complexes with adjacent SecEs at the dimer interface (12, 18).

The Effect of CL on SecYEG Association and Activation of SecA Are Distinct.

The experiments suggest a high-affinity CL binding site on SecYEG that, once occupied, stabilizes the SecYEG dimeric form and creates a high-affinity binding platform for SecA and a conferral of activated ATPase activity. Therefore, we tested if each of the positive effects of CL on SecA (i.e., binding and activation) were the consequence of SecYEG dimerization, in an analysis exploiting the covalently linked SecYEG dimer (11) termed SecYYE2G2.

The protein complexes were first stripped of lipids, then resupplemented with CL, and repurified (Fig. 4A). SecYEG enriched with CL eluted in a volume between the lipid-depleted SecYEG and the covalently linked dimer (Fig. 4A), in agreement with the observations that CL shifts the equilibrium toward dimerization. The respective abilities of SecYEG and SecYYE2G2 to associate with SecA and stimulate the ATPase activity were measured as before (Fig. 4B and Table 3). In contrast to wild-type, the tandem dimer provided a high-affinity binding site for SecA in the absence of CL. In fact, the lipid was only required to achieve a higher rate of ATP turnover (Table 3). Thus, CL operates in two distinct ways: first, on the dimer of SecYEG for the provision of a binding platform for SecA, and second, on the steady-state ATPase activity of SecA.

Fig. 4.
The CL-stabilized SecYEG dimers stimulate the SecA ATPase. (A) 4.2 nmol (8.4 μM) SecYEG with or without 40 μM CL and 4.2 nmol (8.4 μM) tandem linked SecYYE2G2 were applied to a Superose 6 ...
Table 3.
Fitted SecA-SecYEG binding parameters from ATPase binding curves presented in Fig. 4B

CL Does Not Affect the “Inserted” State of SecA.

A fluorescent probe attached at the position 268 of SecY was employed as another measure of the association of SecYEG and SecA (19). The atomic structure of the SecA-SecYEG complex indicates that in the ATP bound state a two-helix finger of SecA inserts into the channel at the exact position of the probe (9), which accounts for an AMP-PNP [adenosine 5′-(β,γ-imido)triphosphate; a nonhydrolyzable analogue of ATP] dependent drop in fluorescence when SecA binds the derivatized complex (SecY268flEG) (19). Therefore, the extrinsic probe monitors an important and well-characterized intermediate of the catalytic cycle.

The fluorescence of SecY268flEG was monitored throughout a titration with SecA in the presence of AMP-PNP and the absence or presence of various lipids. The binding parameters show that CL is the most potent lipid in promoting the association of SecA and SecYEG (Fig. 5 and Table 1). At saturating SecA concentrations, the magnitude of the fluorescence change was not greatly affected by the different lipids. Therefore, in response to the association of SecA and SecYEG the stimulation of ATP turnover requires CL, but the insertion of the two-helix finger does not.

Fig. 5.
Acidic lipids increase the affinity of SecYEG for SecAAMP-PNP but do not affect the insertion of the SecA two-helix finger into SecY. Increasing amounts of SecA were incubated with 29 nM SecY268flEG in detergent solution (0.03% C12E9) containing ...

CL Enables a Nucleotide-Independent Interaction Between SecYEG and SecA.

The high-affinity binding of SecA and SecYEG and dependence on CL was further analyzed by size-exclusion chromatography. SecA and SecYEG do not form a very stable complex in solution (Fig. 6A). In the presence of AMP-PNP there is a significant reduction in the SecA-SecYEG elution volume because of complex formation (Fig. 6A). In the presence of CL, the formation of the SecA-SecYEG complex occurred irrespective of the presence of nucleotide (Fig. 6B). Therefore, CL facilitates the binding of SecA to SecYEG without nucleotides, as it does with the steady-state (ADP) and inserted (ATP) forms of the complex. Therefore, all stages of the hydrolytic cycle are under the influence of CL.

Fig. 6.
Analytical size-exclusion chromatography to monitor the interaction between SecA and SecYEG. We applied 3 nmol SecA (6 μM) and 4.2 nmol (8.4 μM) SecYEG to a Superose 6 HR column in GF buffer, alone and together, ...

Localization of SecYEG Is Affected by the Cardiolipin Content of the Cell.

CL is known to be concentrated at the poles and near the septa of E. coli cells (20); therefore, we tested its influence on the localization of SecYEG. The complex was FlAsH-tagged at the N terminus of SecY or SecE and then expressed at a very low level (without plasmid induction), and their localization was monitored by fluorescence microscopy of intact cells (Fig. 7 A and B and Fig. S3). In the wild-type strain, the complexes were arranged in spiral-like structures in ~30% of the cells, as previously observed (21, 22), and at the poles in ~15% of cells (Fig. 7B and Table S1). The remainder exhibited a uniform distribution. In a cls- strain, which is strongly depleted for CL and augmented in PG, the labelling efficiency was similar to the wild type, but the distribution was reversed, with ~30% of the cells having SecYEG at the poles and only ~15% in spiral structures (Fig. 7B and Table S1).

Fig. 7.
Detection of FlAsH-tagged SecY and SecE in cls+ and cls- strains. (A) Western blot analysis showing that SecY is not overproduced at low concentrations of arabinose in the cls+ (or cls-) strain. Overproduction can be achieved only at high concentration ...

Discussion

SecYEG harbors tightly associated CL, which we show to be important for its conformation and activity. Of the bilayer constituents, the response seems to be specific to CL and to a lesser degree PG. The results explain a number of observations specifying the requirement of acidic phospholipids in protein translocation (15, 23). The inherent symmetry of CL, with two glycerol-linked PGs, might be utilized in the interaction with twin copies of SecYEG. If, as we show, the PG protomer interacts with SecYEG, then both elements of CL could associate with SecYEG to more effectively influence the dimer, presumably through hydrophobic cavities at the interface. An atomic model of the functional translocon on the basis of the SecA-SecYEG structure (9) and the back-to-back form of the SecYEG dimer (12, 18) reveals a space of appropriate dimensions that could accommodate a molecule the size of CL (Fig. 8A). CL has been seen to mediate interactions between the subunits of the mitochondrial ADP/ATP carrier (24) and that of E. coli formate dehydrogenase-N (25) in the manner in which we propose.

Fig. 8.
Modeling of the putative lipid binding site on the membrane bound SecA-(SecYEG)2 complex. (A) Model of the membrane bound translocon (9, 12, 18). The back-to-back SecYEG dimer is represented in space-fill format, with half of the molecule cut away through ...

CL influences the stability of the SecYEG dimer, required for a high-affinity binding platform for SecA as well as the ability to stimulate its ATPase activity. The fused SecYEG dimer proved useful in differentiating these effects, because in this case CL was not required for dimer stabilization. The results show that CL need not directly participate in the binding of SecYEG dimers to SecA. However, its requirement for a high rate of ATP turnover by the SecA-(SecYEG)2 complex is retained. Therefore, the effects of CL on the quaternary structure of SecYEG and affinity for SecA are distinct from those on the ATPase activity. This result is consistent with the observed stimulation of SecA by CL alone (Fig. 2B) (1, 10).

The experiments also show that once SecA is bound to SecYEG, CL has no impact on the ability of the two-helix finger to enter the channel. Therefore, the binding step (promoted by CL-assisted dimerization of SecYEG) must be independent from the downstream conformational transitions at the protein channel. This conclusion is in agreement with the visualization of the inserted state of SecA with monomeric SecYEG, without the obvious contribution of phospholipids (9).

The structure of the SecA-SecYEG complex is such that any CL bound to the membrane section of SecYEG would have to communicate a large distance through the channel and SecA to reach the nucleotide-binding site (9). A study reporting on the lipid-dependent mobility of spin-labeled cysteine residues at the surface of SecA (26) traces a path from the dimer interface of our model (9, 12, 18) to the nucleotide-binding domains (NBDs) (Fig. 8B). A CL molecule at the dimer interface could interact directly with SecA to relay long-distance conformational changes to the NBDs, forcing a change in its hydrolytic cycle. The influence on the quaternary structure (dimer) of SecYEG and on the affinity for SecA, at every stage of the hydrolytic cycle, has profound effects on energy transduction. An uncoupling of ATP hydrolysis from transport results in a reduced ability to translocate preprotein; the reason for this is not yet clear.

The importance of CL for the function and conformation of SecYEG in the cell is also supported by earlier genetic screens. The search for suppressor mutations compensating for the absence of SecG, and poorly active SecA, led to the identification of the phosphatidylglycerophosphate synthase PgsA (2), a key enzyme in the PG and CL synthetic pathway, and also to ScgR, a transcriptional regulator that increases the proportion of CL at the expenses of PG (27), all consistent with the preference for CL over PG observed here.

The importance of acidic lipids to the translocation machinery is reflected by their coincident helical membrane distribution (20) and by the high frequency of protein localization to the cell poles reported here. Because of the four acyl chains and a small head group, CL is classified as “high-curvature lipid” that naturally partitions at the poles and septal regions (28). However, the observation that the CL content influences the distribution of SecYEG to the poles may be indirect. In bacteria, only a few proteins clearly show CL-dependent localization, for example, the osmoregulator ProP (29), yet other proteins with CL-dependent function do not colocalize at the poles (30). The distribution and regulation of protein export and membrane proliferation sites by CL-rich lipid domains is an attractive proposal that warrants further investigation.

The role of CL has mostly been documented in relation to the preservation of optimal activity, stability, and supramolecular organization of bioenergetic enzymes such as the cytochrome bc1 complex (31), ATP synthase (32), cytochrome c oxidase (33), and the photoreaction center (34). Even after extensive purification, these enzymes retain bound CL, and complete delipidation consistently leads to the irreversible dissociation of their native oligomeric structures (33, 34). Our results provide a comprehensive description of an active role for CL in Sec-dependent protein transport. These special dependencies may also hold true in translocation systems found in other energy-conserving membranes, e.g., in mitochondria. Clearly, there is an important and common role for this phospholipid across multiple energy-transducing systems.

Materials and Methods

Chemicals and Biochemicals.

Lipids were purchased from Avanti Polar Lipids and DDM (n-dodecyl-β-D-maltoside) from Anatrace, the EnzChek kit and DAPI (4,6-diamidino-2-phenylindole) were supplied by Invitrogen, chromatographic columns were acquired from GE Healthcare, and the TLC plates from Merck. All other reagents were acquired from Sigma, and FLAsH-EDT2 was synthesized as described (35).

Overexpression and Purification of Protein Translocation Components.

Wild-type SecA, SecYEG, and SecYK268CEG were produced and purified as before (10, 19). The genetically fused SecY dimer (SecYYE2G2) (13), encoded by plasmid pBAD-EHis6YYG, was produced in c43 cells (36), grown to an OD600 of 1 in a 30-liter Bio Bench bioreactor (Applikon Biotechnology). Cells were harvested 3.5 h after promoter induction with 0.8% (wt/vol) arabinose and the complex purified as for SecYEG. proOmpA Δ176-296 Strep was overexpressed and purified as described (15). Protein quantification was determined by using extinction coefficients of 99,000 M-1 cm-1 (SecA), 139,000 M-1 cm-1 (SecYEG), and 49,860 M-1 cm-1 (proOmpA Δ176-296 Strep).

[32P]-labeling of membranes was achieved by using E. coli cells growing in LB medium containing [32P]orthophosphate (1 μC/mL of culture) overproducing the His-tagged SecYEG complex (1 h at 37 °C with 0.5% arabinose). The cells were lysed by lysozyme/EDTA/cold-water treatment and the membranes prepared as described (37). The SecYEG complex was isolated as before (38), and lipids were extracted and analyzed by TLC as described in SI Text.

Delipidation and Relipidation of the SecYEG Complex.

Preparation of lipid stocks is described in SI Text. SecYEG was lipid-depleted during the extraction and purification. The standard purification procedure employed 0.1% C12E9 during Ni2+-affinity chromatography and 0.05% during gel filtration (38). A more stringent protocol was adopted, respectively using 0.5% and 0.1% C12E9. The lipid-depleted sample was incubated overnight at 4 °C with a 10-fold molar excess of either CL or PL, also dissolved in C12E9. Unbound lipids were removed by gel filtration (Superdex 200 XK 16/60) equilibrated in 20 mM Tris-Cl pH 8.0, 130 mM NaCl, 0.05% (wt/vol) C12E9. The lipid-depleted SecYEG sample was reconstituted into liposomes in a way that has been described (38). Proportions of the individual lipids used to make liposomes of dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG)/CL were 67%, 23%, and 10%, respectively, to match that of the E. coli PL mixture. In the absence of CL, this was altered to 72% DOPE and 28% DOPG. Lipids were extracted and analyzed by TLC as described in SI Text.

Steady-State ATPase Assays and in Vitro Translocation.

Steady-state ATPase assays and translocation of proOmpA into proteoliposomes were carried out as before (16) and are described in SI Text.

BN-PAGE analysis.

Purification and [125I]-labeling of SecYEG (to ~1.5 × 106 cpm/μg) were performed in DDM (as indicated) and TSG buffer (25 mM Tris-Cl, pH 7.5; 50 mM NaCl; 10% glycerol) and BN-PAGE was carried out as before (39) and is described in SI Text.

Photoinduced Cross-Linking of Unmodified Proteins.

A lipid-depleted SecYEG preparation was cross-linked in the presence of 40 μM PL, PE, PG, or CL by using 2 mM ammonium persulphate and 0.1 mM Tris-bipyridylruthenium(II) (17, 19). After irradiation for 45 s, the reaction was quenched with 0.1 M DTT and the cross-linked products were identified by SDS-PAGE and Western blotting.

Analytical Size-Exclusion Chromatography.

SecA (3 nmol), SecYEG, or SecYYE2G2 (4.2 nmol) were applied onto a Superose 6 HR column equilibrated in GF buffer [20 mM Tris-Cl pH 8, 0.13 M NaCl, 2 mM MgCl2, 10% (vol/vol) glycerol, 0.03% (wt/vol) C12E9] with or without 40 μM CL. Where indicated, the AMP-PNP concentration was 1 mM in the incubation buffer and 100 μM in the elution buffer.

Measurement of a Conformational Change in SecYEG by Fluorescence Spectroscopy.

The SecYEG complex with the mutation K268C, in an otherwise cysteineless background, was labeled with 5-iodoacetamidofluorescein to produce SecY268flEG, which was monitored as previously described (19) and is described in SI Text.

Protein Localization by Fluorescence Microscopy.

Plasmid pBAD22-FlashY and pBAD22-FlashE, encoding for SecYEG with a FlAsH-tag (sequence CCPGCC) at the N terminus of SecY or SecE, respectively, are derived from pBAD22-EYG (24). Plasmid pBAD22-FlashY rescues the growth of strain CJ107 (secY42ts) on M9 media, with or without induction with arabinose, and in vitro translocation assays show that the FlAsH-tag does not compromise the SecYEG activity. The plasmids were introduced into E. coli strains WG350 (cls+) and WG980 (cls-) (29). Protein labelling and localization by fluorescence microscopy is described in SI Text.

Supplementary Material

Supporting Information:

Acknowledgments.

We are grateful to Profs. Tony Clarke and Janet Wood for discussion of this work, Sir John Walker for the c43 strains of E. coli, Dr. Dilem Hizlan for preliminary TLC analysis, and Dr. Ryan Schulze for critical reading of this manuscript. T.R. analyzed the subcellular localization of the Sec complex in the laboratory of Dr. Janet M. Wood with support from Discovery Grant OPG0000508 from the Natural Sciences and Engineering Research Council of Canada. The SecA-(SecYEG)2 model was built by Dr. Richard Sessions, the details of which will be available in a future publication. This work was funded by the Canadian Institute of Health Research (F.D.), the Wellcome Trust [Grants 084452 and 082140 (to I.C.)], and the Biotechnology and Biological Sciences Research Council [BB/F002343/1 (to I.C.)].

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0914680107/-/DCSupplemental.

References

1. Lill R, Dowhan W, Wickner W. The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell. 1990;60:271–280. [PubMed]
2. Suzuki H, Nishiyama K, Tokuda H. Increases in acidic phospholipid contents specifically restore protein translocation in a cold-sensitive secA or secG null mutant. J Biol Chem. 1999;274:31020–31024. [PubMed]
3. Hendrick JP, Wickner W. SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane. J Biol Chem. 1991;266:24596–24600. [PubMed]
4. de Vrije T, de Swart R, Dowhan W, Tommassen J, de Kruijff B. Phosphatidylglycerol is involved in protein translocation across Escherichia coli inner membranes. Nature. 1988;334:173–175. [PubMed]
5. Alami M, Dalal K, Lelj-Garolla B, Sligar SG, Duong F. Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA. EMBO J. 2007;26:1995–2004. [PMC free article] [PubMed]
7. Benach J, et al. Phospholipid-induced monomerization and signal-peptide-induced oligomerization of SecA. J Biol Chem. 2003;278:3628–3638. [PubMed]
8. Or E, Navon A, Rapoport TA. Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane. EMBO J. 2002;21:4470–4479. [PMC free article] [PubMed]
9. Zimmer J, Nam Y, Rapoport TA. Structure of a complex of the ATPase SecA and the protein-translocation channel. Nature. 2008;455:936–943. [PubMed]
10. Gold VA, Robson A, Clarke AR, Collinson I. Allosteric regulation of SecA: Magnesium-mediated control of conformation and activity. J Biol Chem. 2007;282:17424–17432. [PubMed]
11. Tam PC, Maillard AP, Chan KK, Duong F. Investigating the SecY plug movement at the SecYEG translocation channel. EMBO J. 2005;24:3380–3388. [PMC free article] [PubMed]
12. Breyton C, Haase W, Rapoport TA, Kühlbrandt W, Collinson I. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature. 2002;418:662–665. [PubMed]
13. Duong F. Binding, activation and dissociation of the dimeric SecA ATPase at the dimeric SecYEG translocase. EMBO J. 2003;22:4375–4384. [PMC free article] [PubMed]
14. Tziatzios C, et al. The bacterial protein-translocation complex: SecYEG dimers associate with one or two SecA molecules. J Mol Biol. 2004;340:513–524. [PubMed]
15. Osborne AR, Rapoport TA. Protein translocation is mediated by oligomers of the SecY complex with one SecY copy forming the channel. Cell. 2007;129:97–110. [PubMed]
16. Robson A, Gold VA, Hodson S, Clarke AR, Collinson I. Energy transduction in protein transport and the ATP hydrolytic cycle of SecA. Proc Natl Acad Sci USA. 2009;106:5111–5116. [PMC free article] [PubMed]
17. Fancy DA, Kodadek T. Chemistry for the analysis of protein-protein interactions: Rapid and efficient cross-linking triggered by long wavelength light. Proc Natl Acad Sci USA. 1999;96:6020–6024. [PMC free article] [PubMed]
18. Bostina M, Mohsin B, Kuhlbrandt W, Collinson I. Atomic model of the E. coli membrane-bound protein translocation complex SecYEG. J Mol Biol. 2005;352:1035–1043. [PubMed]
19. Robson A, Booth AE, Gold VA, Clarke AR, Collinson I. A large conformational change couples the ATP binding site of SecA to the SecY protein channel. J Mol Biol. 2007;374:965–976. [PubMed]
20. Barak I, Muchova K, Wilkinson AJ, O’Toole PJ, Pavlendova N. Lipid spirals in Bacillus subtilis and their role in cell division. Mol Microbiol. 2008;68:1315–1327. [PMC free article] [PubMed]
21. Campo N, et al. Subcellular sites for bacterial protein export. Mol Microbiol. 2004;53:1583–1599. [PubMed]
22. Shiomi D, Yoshimoto M, Homma M, Kawagishi I. Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery. Mol Microbiol. 2006;60:894–906. [PMC free article] [PubMed]
23. Kusters R, Dowhan W, de Kruijff B. Negatively charged phospholipids restore prePhoE translocation across phosphatidylglycerol-depleted Escherichia coli inner membranes. J Biol Chem. 1991;266:8659–8662. [PubMed]
24. Nury H, et al. Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers. FEBS Lett. 2005;579:6031–6036. [PubMed]
25. Jormakka M, Tornroth S, Byrne B, Iwata S. Molecular basis of proton motive force generation: Structure of formate dehydrogenase-N. Science. 2002;295:1863–1868. [PubMed]
26. Cooper DB, et al. SecA, the motor of the secretion machine, binds diverse partners on one interactive surface. J Mol Biol. 2008;382:74–87. [PMC free article] [PubMed]
27. Kontinen VP, Helander IM, Tokuda H. The secG deletion mutation of Escherichia coli is suppressed by expression of a novel regulatory gene of Bacillus subtilis. FEBS Lett. 1996;389:281–284. [PubMed]
28. Mukhopadhyay R, Huang KC, Wingreen NS. Lipid localization in bacterial cells through curvature-mediated microphase separation. Biophys J. 2008;95:1034–1049. [PMC free article] [PubMed]
29. Romantsov T, et al. Cardiolipin promotes polar localization of osmosensory transporter ProP in Escherichia coli. Mol Microbiol. 2007;64:1455–1465. [PubMed]
30. Romantsov T, Battle AR, Hendel JL, Martinac B, Wood JM. Protein localization in Escherichia coli cells: Comparison of cytoplasmic membrane proteins ProP, LacY, ProW, AqpZ, MscS, and MscL. J Bacteriol. 2010;192(9):2471. [PMC free article] [PubMed]
31. Zhang M, Mileykovskaya E, Dowhan W. Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria. J Biol Chem. 2005;280:29403–29408. [PMC free article] [PubMed]
32. Wittig I, Schagger H. Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes. Biochim Biophys Acta. 2009;1787:672–680. [PubMed]
33. Dowhan W. Molecular basis for membrane phospholipid diversity: Why are there so many lipids? Annu Rev Biochem. 1997;66:199–232. [PubMed]
34. McAuley KE, et al. Structural details of an interaction between cardiolipin and an integral membrane protein. Proc Natl Acad Sci USA. 1999;96:14706–14711. [PMC free article] [PubMed]
35. Adams SR, Tsien RY. Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins. Nat Protoc. 2008;3:1527–1534. [PMC free article] [PubMed]
36. Miroux B, Walker J. Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol. 1996;260:289–298. [PubMed]
37. Joly JC, Leonard MR, Wickner WT. Subunit dynamics in Escherichia coli preprotein translocase. Proc Natl Acad Sci USA. 1994;91:4703–4707. [PMC free article] [PubMed]
38. Collinson I, et al. Projection structure and oligomeric properties of a bacterial core protein translocase. EMBO J. 2001;20:2462–2471. [PMC free article] [PubMed]
39. Bessonneau P, Besson V, Collinson I, Duong F. The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure. EMBO J. 2002;21:995–1003. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...