Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane - PubMed (original) (raw)
Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane
Eran Or et al. EMBO J. 2002.
Abstract
The ATPase SecA mediates post-translational translocation of precursor proteins through the SecYEG channel of the bacterial inner membrane. We show that SecA, up to now considered to be a stable dimer, is actually in equilibrium with a small fraction of monomers. In the presence of membranes containing acidic phospholipids or in certain detergents, SecA completely dissociates into monomers. A synthetic signal peptide also affects dissociation into monomers. In addition, conversion into the monomeric state can be achieved by mutating a small number of residues in a dimeric and fully functional SecA fragment. This monomeric SecA fragment still maintains strong binding to SecYEG in the membrane as well as significant in vitro translocation activity. Together, the data suggest that the SecA dimer dissociates during protein translocation. Since SecA contains all characteristic motifs of a certain class of monomeric helicases, and since mutations in residues shared with the helicases abolish its translocation activity, SecA may function in a similar manner.
Figures
Fig. 1. Steady-state FRET between coumarin- and fluorescein-labeled SecA molecules. Coumarin- and fluorescein-labeled SecA heterodimers (dark gray traces) are compared with coumarin–SecA/unlabeled SecA (black traces) and fluorescein–SecA/unlabeled SecA (light gray traces) heterodimers. The following additions were made: (A) none; (B) 36 µg of unlabelled SecA; (C) 52 µg of BSA; (D) 24 µg of monomeric N68-SecA; (E) 5 µl of liposomes containing E.coli phospholipids; (F) 5 µl of DOPE/DOPG liposomes; (G) 5 µl of DOPE/DOPC liposomes; (H) 1.5 mg/ml ddm.
Fig. 1. Steady-state FRET between coumarin- and fluorescein-labeled SecA molecules. Coumarin- and fluorescein-labeled SecA heterodimers (dark gray traces) are compared with coumarin–SecA/unlabeled SecA (black traces) and fluorescein–SecA/unlabeled SecA (light gray traces) heterodimers. The following additions were made: (A) none; (B) 36 µg of unlabelled SecA; (C) 52 µg of BSA; (D) 24 µg of monomeric N68-SecA; (E) 5 µl of liposomes containing E.coli phospholipids; (F) 5 µl of DOPE/DOPG liposomes; (G) 5 µl of DOPE/DOPC liposomes; (H) 1.5 mg/ml ddm.
Fig. 2. Exchange of subunits between FLAG- and His6-tagged SecA. In the standard procedure (lane 1), the differently tagged SecA samples were mixed and the His-tagged proteins were recovered with Ni-NTA beads. The bound proteins were separated by SDS–PAGE and analyzed by immunoblotting with anti-FLAG and anti-His antibodies. In lane 2, SecA rather than SecA-His6 was added. For the sample in lane 3, the beads were washed with 140 mM imidazole before analysis of the bound material. In lanes 4 and 5, 2.5 mM MgCl2 and 2.5 mM MgCl2 plus 1.5 mM ATP, respectively, were added. For the sample in lane 6, 2 mg/ml ddm was present during the incubation.
Fig. 3. Cross-linking of SecA with the bifunctional cross-linker EDAC. (A) The samples were separated by SDS–PAGE and stained with Coomassie Blue. The following additions were made: lane 4, 2 mM ATP; lane 5, 2 mM ADP; lane 6, 2 mM ATPγS; lane 7, 7.5 µl of E.coli liposomes with reconstituted SecYEG; lane 8, 7.5 µl of liposomes made from E.coli phospholipids; lane 9, 7.5 µl of DOPE/DOPG liposomes; lane 10, 7.5 µl of DOPE/DOPC liposomes; lane 11, 3 mg/ml ddm. The control in lane 2 received glycine and sample buffer before EDAC. (B) Cross-linking of [125I]SecA with EDAC. Samples were resolved by SDS–PAGE and analyzed by autoradiography. The following additions were made: lanes 1 and 2, 7.5 µg of SecA; lanes 5–8, 2 µl of E.coli liposomes with reconstituted SecYEG; lane 6, 2 mM ATP; lane 7, 2 mM ADP; lane 8, 2 mM AMP–PNP. Samples 1 and 3 are controls and received glycine and sample buffer before EDAC.
Fig. 4. Analysis of SecA and of SecA derivatives by sucrose density centrifugation. The samples contained: (A) 9 µg of SecA; (B) 9 µg of SecA mixed with 0.15% ddm, run in a gradient containing 0.05% ddm; (C) 17 µg of N95-SecA; (D) 17 µg of N95-SecA-3Ala; (E) 17 µg of N95-SecA-6Ala. All samples also contained 50 µg of BSA. Samples (90 µl) from fractions 1–20 were analyzed by SDS–PAGE and stained with Coomassie Blue.
Fig. 5. A signal peptide dissociates SecA dimers. SecA in 60 µl buffer containing 4% DMSO was treated with EDAC in the presence of increasing concentrations of the synthetic wild-type signal peptide KRR-LamBWT or the mutant peptide KRR-LamBΔ78 (Alpha Diagnostic Int.). Samples were analyzed by SDS–PAGE and stained with Coomassie Blue. For a control, glycine and sample buffer were added before EDAC.
Fig. 6. Native gel electrophoresis of SecA and SecA derivatives. Four micrograms of SecA, 3 µg of N95-SecA, 3 µg of N95-SecA-3Ala or 3 µg of N95-SecA-6Ala were resolved in a native gel. The positions of the size standards catalase (230 kDa) and BSA (68 kDa) are indicated. An asterisk marks the position of misfolded N95-SecA-6Ala.
Fig. 7. (A) A monomeric derivative of SecA retains strong interaction with SecYEG. Binding of [125I]SecA (50 nM) to proteoliposomes containing reconstituted SecYEG was performed in the presence of 0–4 µM SecA, 0–4.5 µM N95-SecA or 0–4.5 µM N95-SecA-6Ala. Non-specific binding, which was 16% of total binding, was subtracted. The specific binding of [125I]SecA in the absence of competitor was taken as 100%. (B) A monomeric derivative of SecA retains significant translocation activity. Translocation of [35S-Met]proOmpA into reconstituted proteoliposomes was tested with full-length SecA and a monomeric SecA fragment (N95-SecA-6Ala). The reconstituted proteoliposomes contained either wild-type SecYEG complex (rec.YEG) or a mutant complex containing the prlA4 mutation in SecY [rec.Y(prlA4)EG]. Where indicated, ATP was present during the reaction. All samples were treated with proteinase K. Where indicated, Triton X-100 was present during proteolysis. Lane 1 shows 15% of input [35S-Met]proOmpA. (C) Kinetics of translocation of [35S-Met]proOmpA into proteoliposomes containing reconstituted SecY(PrlA4)EG was followed in the presence of either SecA or N95-SecA-6Ala at 25°C. Protease-protected proOmpA were quantitated using a phosphoimager.
Fig. 8. Residues conserved between SecA and monomeric helicases are important for SecA translocation ATPase activity. Wild-type SecA, SecA(Thr504Ala) and SecA(Arg574Ala) were tested for ATPase activity in the presence of proteoliposomes containing reconstituted SecYEG (+rec.YEG) or in the absence of liposomes (–rec.YEG). All samples also contained proOmpA.
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