Maximal efficiency of coupling between ATP hydrolysis and translocation of polypeptides mediated by SecB requires two protomers of SecA

Abstract

SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in Escherichia coli during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.

FIG. 1.

Translocation of pro-OmpA mediated by A1:B4 and A2:B4 complexes. (A) Protection of pro-OmpA; (B) associated ATPase activity of the translocation. In vitro assay mixtures contained pro-OmpA, SecA species, and SecB species at 2 μM each, expressed as dimeric SecA and tetrameric SecB, as follows: wild-type SecA (circles), SecAdN7 (down triangles), and SecAdN10 (squares) with wild-type SecB; SecAC4 with wild-type SecB (up triangles); and wild-type SecA with SecBL75Q (diamonds). The data here and in Fig. 2 and 3 were fitted to a hyperbola using SigmaPlot 2001 software. The error bars show standard deviations. All assays were done at least three times. Where error bars are not obvious, they fall within the symbols. For all fits, 0.45 min was used as the intercept on the x axis.

FIG. 2.

Translocation of precursor galactose-binding protein mediated by A1:B4 and A2:B4 complexes. (A) Protection of precursor galactose-binding protein (pGBP); (B) associated ATPase activity of the translocation. Concentrations of proteins and symbols are the same as in Fig. 1. The samples were analyzed by immunoblotting.

FIG. 3.

Translocation of pro-OmpA in the presence and absence of SecB. In vitro translocation of 2 μM pro-OmpA by wild-type SecA (circles) or SecAdN7 (triangles) at 2 μM SecA expressed as a dimer was carried out in the presence (filled symbols) or absence (open symbols) of 2 μM SecB expressed as a tetramer. The efficiency of translocation without SecB is extremely low; therefore, to maximize activity for these experiments, 1.7 mM NADH, 7.5 mM phosphocreatine, and 37 mg/ml creatine phosphokinase were included in the in vitro system to regenerate ATP. The samples were analyzed by immunoblotting.

FIG. 4.

Cross-linking of AET-SecA to precursors. (A) Cross-linking to pro-OmpA; (B) cross-linking to precursor galactose-binding protein (pGBP). Immunoblots using antiserum to SecA are shown in the upper panels, and those using antisera to the precursors are in the lower panels. Cross-linked complexes are indicated by dots and irradiation by hν. Molecular mass markers (MW) are shown on the right.