13190 Biochemistry 1993,32, 13190-13 197 SecA, the Peripheral Subunit of the Escherichia coli Precursor Protein Translocase, Is Functional as a Dimer? Arnold J. M. Driessen’ Department of Microbiology, University of Groningen. Kerklaan 30, 9751 NN Haren, The Netherlands Received June 28, 1993; Revised Manuscript Received August 30, 1993” ABSTRACT: SecA, the peripheral ATPase domain of the Escherichia coli precursor protein translocase, was denatured in 6 M guanidine hydrochloride. Circular dichroism and intrinsic tryptophan fluorescence spectra revealed that the protein is transformed into a random-coil configuration. Upon dilution of the chaotropic agent, SecA refolds into its native, functional conformation as a homodimer. As structural criteria, the native dimeric state was assayed by size-exclusion chromatography, chemical cross-linking, tryptophan fluorescence, and circular dichroism. Functional SecA heterodimers were formed of which the individual subunits were tagged with fluorescent dyes to allow measurements of the association state of the monomers by resonance energy transfer using steady-state and time-resolved fluorescence spectroscopy. SecA retained its dimeric structure during translocation, while energy transfer was abolished only by denaturation. The “half-of-the-sites activity” was investigated by constructing heterodimers formed from native and &azido- ATP-inactivated SecA. Heterodimers have lost the ability to support translocation of the precursor protein proOmpA in an in uitro translocation system. It is concluded that the dimeric structure is maintained during translocation and required for functionality. SecA (Schmidt et al., 1988) is a dissociable, peripheral subunit of the precursor protein translocase of Escherichia coli (Brundage et al., 1990; Hartl et al., 1990; Wickner et al., 1991). Inaddition toSecA, translocaseconsistsoftheSecY/E protein, a multisubunit integral membrane protein complex (Brundage et al., 1990, 1992; Akimura et al., 1991), and possibly SecD and SecF (Bieker-Brady & Silhavy, 1992; Matsuyama et al., 1993). SecA is an ATPase, and the concerted activities of the protonmotive force and ATP hydrolysis by SecA permit the successive progress of precursor proteins across the membrane (Tani et al., 1989,1990; Schiebel et al., 1991; Driessen, 1992a; Arkowitz et al., 1993). At the transitional stages of translocation, the precursor protein may physically interact with the SecA and SecY subunits of the translocase (Joly & Wickner, 1993). SecA is a complex protein and involved in multiplecatalytic and regulatory interactions. SecA interacts with (i) the signal sequence domain and as yet unknown elements of the mature domain of precursor proteins (Lill et al., 1990; Akita et al., 1990; Joly & Wickner, 1993), (ii) the SecB protein, thereby assisting in protein targeting (Hartl et al., 1990), (iii) the SecY/Eprotein at the membrane surface (Hartl et al., 1990), (iv) acidic phospholipids (Lill et al., 1991), and (v) its own mRNA at a sequencearound the gene X-secA intergenic region (Dolan & Oliver, 1991) as part of an autogenous regulation mechanism. Interactions with translocation-competentpre- cursor proteins, the SecY/E protein, and acidic phospholipids (Lill et al., 1989, 1990; Cunningham & Wickner, 1989; Brundage et al., 1990) activate SecA for ATP hydrolysis. Binding of ATP to SecA drives the translocation of the amino terminus of the precursor protein, while ATP hydrolysis is needed to release the precursor protein from its association with SecA (Schiebel et al., 1991; Driessen, 1992b). +This work was supported by the Netherlands Organization for Scientific Research (NWO). * Address correspondence to this author. Phone: +50-62 21 64. Fax: +50-63 52 05. E-mail (Internet): DRIESSEN@BIOL.RUG.NL. * Abstract published in Advance ACS Abstracts, November 15,1993. 0006-2960/93/0432-13 190$04.00/0 The number of SecA molecules present in a typical E. coli cell is about 10-fold higher than that of the other components of the translocase (Matsuyama et al., 1992). Localization studies indicate a dynamic and complex behavior of the cellular SecA. Depending on the conditions of cell fractionation, up to half of the cellular SecA can be found to be associated with the cytoplasmic membrane, while the remainder is present in a soluble fraction (Akita et al., 1991; Cabelli et al., 1991). A small amount of the cytosolic SecA appears to be present in a labile high molecular mass complex, Le., 500-1000 kDa, with unknown composition. Size-exclusion chromatography, velocity sedimentation analysis, and chemical cross-linking experimentshave shown that cytosolic SecA, and SecA purified from thecytosolic fraction, is homodimeric (Akita et al., 1991; Cabelli et al., 1991; van der Wolk et al., 1993). The dimeric state of the purified SecA is maintained over a wide range of protein concentrations (Akita et al., 1991). SecA homodimers can be functionallyreconstituted from the guanidinedenatured state, and during renaturation, reducing conditions which prevented disulfidebond formation were found not to be critical for dimerization. Both in vivo (Cabelli et al., 1991; Kusters et al., 1992) and in vitro (Breukink et al., 1992; van der Wolk et al., 1993) studies suggest that the cellular location of SecA is modulated by its interaction with nucleotides. Genetic studies are consistent with the suggestion that SecA is a dissociable component of the translocase, arguing that the components of the translocase assemble and disassemble during translocation (Bieker-Brady & Silhavy, 1992). No data are available on the quaternary structure of SecA during the various stages of translocation, and the question arises as to whether the aggregation state of SecA is modulated by interactions with the various ligands. Also, the functional consequence of the dimeric organization of the soluble SecA had remained obscure. The dimeric structure of SecA raises the question as to whether the two identical polypeptide chains constitute a single functional unit. As recently indicated (Weaver et al., 1992), genetic data are consistent with a homomultimeric association state of SecA. Dominant-negative secA mutations are 0 1993 American Chemical Society