Comparative characterization of SecA from the alpha-subclass purple bacterium Rhodobacter capsulatus and Escherichia coli reveals differences in membrane and precursor specificity

Author:

Helde R1,Wiesler B1,Wachter E1,Neubüser A1,Hoffschulte H K1,Hengelage T1,Schimz K L1,Stuart R A1,Müller M1

Affiliation:

1. Adolf Butenandt Institut für Physikalische Biochemie, Ludwig-Maximilians-Universität München, Munich, Germany.

Abstract

We have cloned the secA gene of the alpha-subclass purple bacterium Rhodobacter capsulatus, a close relative to the mitochondrial ancestor, and purified the protein after expression in Escherichia coli. R. capsulatus SecA contains 904 amino acids with 53% identity to E. coli and 54% identity to Caulobacter crescentus SecA. In contrast to the nearly equal partitioning of E. coli SecA between the cytosol and plasma membrane, R. capsulatus SecA is recovered predominantly from the membrane fraction. A SecA-deficient, cell-free synthesis-translocation system prepared from R. capsulatus is used to demonstrate translocation activity of the purified R. capsulatus SecA. This translocation activity is then compared to that of the E. coli counterpart by using various precursor proteins and inside-out membrane vesicles prepared from both bacteria. We find a preference of the R. capsulatus SecA for the homologous membrane vesicles whereas E. coli SecA is active with either type of membrane. Furthermore, the two SecA proteins clearly select between distinct precursor proteins. In addition, we show here for the first time that a bacterial c-type cytochrome utilizes the canonical, Sec-dependent export pathway.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

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