Topology Inversion of SecG Is Essential for Cytosolic SecA-dependent Stimulation of Protein Translocation

Abstract

SecG, a subunit of the protein translocon, undergoes a cycle of topology inversion. To further examine the role of this topology inversion, we analyzed the activity of membrane vesicles carrying a SecG-PhoA fusion protein (SecG-PhoA inverted membrane vesicles (IMVs)). In the absence of externally added SecA, SecG-PhoA IMVs were as active in protein translocation as SecG ؉ IMVs per SecA. Consistent with this observation, insertion of membrane-bound SecA into SecG-PhoA IMVs was normally observed. On the other hand, externally added SecA did not affect the activity of SecG-PhoA IMVs, but it caused >10-fold stimulation of the translocation activity of SecG ؉ IMVs, indicating that the topology inversion of SecG, which cannot occur in SecG-PhoA IMVs, is essential for cytosolic SecA-dependent stimulation of protein translocation. SecG-PhoA IMVs generated a 46-kDa fragment of SecA upon trypsin treatment. The accumulation of this membrane-inserted SecA in the SecGPhoA IMVs was responsible for the loss of the soluble SecA-dependent stimulation. Moreover, fixation of the inverted SecG topology was found to be dependent on soluble SecA. The dual functions of SecG in protein translocation will be discussed.Most secretory proteins of Escherichia coli are synthesized with an N-terminally extended signal sequence and then posttranslationally translocated across the cytoplasmic membrane via the SecYEG translocon using the driving force provided by translocation ATPase SecA (see recent reviews in Refs. 1 and 2). SecA plays a central role in protein translocation by interacting with many ligands, including presecretory proteins, membrane lipids, SecYEG, and SecDF, and it undergoes a dynamic structure change coupled with protein translocation. SecA, which can be purified as a soluble protein, is inserted deep into the membrane upon the binding of both ATP and a precursor protein, which causes protein translocation of a segment of 20 -30 amino acids (3-5). Thus the translocation of the precursor is thought to be driven by the repeat of the membrane insertiondeinsertion cycle of SecA and to proceed in a stepwise manner. Based on the crystal structures of the translocon and SecA (see Refs. 1 and 2, and references therein), detailed mechanisms of protein translocation are proposed; however, they are derived from a limited number of snapshots of the catalytic cycle. Therefore, many issues, including the oligomeric state of each subunit, structure changes upon protein translocation, mode of action of the translocon, and so on, remain to be clarified.The SecG subunit of the SecYEG translocon possesses two transmembrane stretches with N and C termini exposed to the periplasm (6, 7). We have reported that SecG undergoes a cycle of topology inversion, which couples the SecA cycle with protein translocation (7-10). These phenomena were demonstrated by the translocation-dependent changes in proteinase K (PK) 2 sensitivity of the C-terminal region of SecG and by the chemical labeling of the cysteine-containing SecG mutants, using in vitro and i...

Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation

“…We studied the significance of SecG inversion by means of IMV and mutant cells in which SecYEG in either the WT or a mutant form had been expressed at the WT level (9,(17)(18)(19)(20). Under the conditions used, MPIase could sufficiently interact with SecYEG.…”

“…The major difference between this report and our study is that inverted membrane vesicles (IMV) either with an overproduced amount (23) or the WT level (our study) of SecYEG were used. Later, we found that SecG inversion cannot be reproduced under SecYEG overproduction conditions (18,23). Therefore, it is suggested that a factor or factors other than known Sec factors is required to invert SecG, as the factor should be limiting on SecYEG overproduction.…”

“…One of the most dynamic structural changes of the translocon so far proposed is the topology inversion cycle of SecG (17)(18)(19)(20). In the absence of preprotein translocation, both the N-and Cterminal regions are exposed to the periplasmic space (Fig.…”

“…Soluble SecA is also important for SecG inversion (18,19). SecG inversion is supported by three independent lines of evidence; namely, (i) preprotein translocation is inhibited by an anti-SecG antibody that recognizes the periplasmic region of SecG when added from the cytoplasmic side (17,22), (ii) proteinase K digests SecG on the opposite side of membranes when preprotein translocation occurs (9,(17)(18)(19), and (iii) membrane-impermeable reagents react with SecG regions on the opposite side of membranes on preprotein translocation (20). However, a report that disfavors this model of SecG inversion stated that SecG, crosslinked to SecY through Cys residues introduced into both proteins at appropriate positions, is fully functional (23).…”

Glycolipozyme membrane protein integrase (MPIase): recent data

“…However, when each SecYEG with a Cys residue was reconstituted together with MPIase, the SecG dimer but not the SecE dimer was efficiently formed, indicating that MPIase transformed the back-to-back dimer into a side-by-side dimer ( Figure 5) (46). It has been reported that the SecG subunit of SecYEG, which significantly stimulates preprotein translocation (51-53), undergoes a topology inversion cycle coupled with preprotein translocation (54)(55)(56)(57). It has also been demonstrated that this remarkable structure change of SecG is essential for the SecG function (54,56).…”