Interaction between SecA and SecYEG in Micellar Solution and Formation of the Membrane-Inserted State

Abstract

Preprotein translocation in Escherichia coli is mediated by the translocase with SecA as peripheral ATPase and SecY, SecE, and SecG as membrane domain. To facilitate large-scale purification of the SecYEG heterotrimer, SecY was fused at its amino terminus with a hexahistidine tag and co-overexpressed with SecE and SecG. The presence of the His tag allowed purification of homogeneously pure SecYEG complex by a single anion-exchange chromatographic step starting from octyl glucoside-solubilized inner membranes. Endogenous levels of SecD and SecF copurified with the SecYEG protein. Purified SecYEG complex retained a nativelike, alpha-helical conformation in octyl glucoside and in micellar solution binds SecA with high affinity. In the presence of the nonhydrolyzable nucleotide analogue adenosine 5'-(beta, gamma-imidotriphosphate), octyl glucoside-solubilized SecYEG is nearly as effective as the reconstituted enzyme in inducing the formation of a proteinase K-protected 30 kDa fragment of 125I-labeled SecA, while SecYEG is proteolyzed to fragments smaller than 6 kDa. These data demonstrate that the 30-kDa SecA fragment is not protected by the lipid phase nor by SecYEG but rather indicate that it represents a SecYEG- and nucleotide-induced stable conformational state of a SecA domain.

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

“…Since the Glu176 mutants of SecY are disturbed in supporting 30 kDa formation of SecA and not in SecA binding, we hypothesize that this conformational change is induced by "membrane insertion" of SecA, and thus that SecA only interacts with the TMS4c region in the "inserted" state. Although the extent of membrane insertion of SecA as originally proposed 17 is questionable, 36 TMS4c could be part of the membrane region where SecA actually inserts.…”

Demonstration of a Specific Escherichia coli SecY−Signal Peptide Interaction

“…When AMP-PCP is present, SecA is in a more extended conformation (58, 59) and we observe higher levels of signal peptide-SecA cross-linking. Together with SecYEG, this may represent a SecA inserted state of the translocase (44) and corresponds to the initial delivery of the signal peptide into the membrane. Hydrolysis of ATP yields the more compact ADP-bound form of SecA (58,59).…”

“…SecY was identified after Western blotting using conventional procedures and anti-SecY antibody (1:2000), a generous gift of W. Wickner, Dartmouth Medical School. DEAE-52 purified His (6) -SecEYG was reconstituted into proteoliposomes as described (44)(45)(46).…”

The ATPase domain of SecA can form a tetramer in solution 1 1Edited by I. B. Holland

“…13,14 Each cycle of ATP binding and hydrolysis results in translocation of approximately 5 kDa of preprotein, 15,16 and therefore repeated cycles of ATP hydrolysis would be required to translocate the entire preprotein. In this model, ATP binding 17 is thought to initiate profound conformational changes in SecA, such that most of the SecA molecule becomes resistant to proteolysis 14,18 and accessible from the periplasmic side of the translocon. 19 ± 21 Data on the structure and composition of the SecA-SecYEG complex need to be integrated with this model of translocation.…”