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

Abstract

We have cloned the secA gene of the ␣-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 synthesistranslocation 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.

The Putative Assembly Factor CcoH Is Stably Associated with the cbb 3 -Type Cytochrome Oxidase

“…T7 RNA polymerase-dependent in vitro expression of ccoH was achieved using plasmid pET19b-ccoH. Cell-free protein synthesis using [ 35 S]methionine and cysteine with R. capsulatus S-135 extracts was carried out for 30 min at 35°C as described previously (15,26,55). For cotranslational integration of in vitrosynthesized proteins into membranes, ICMs were added after 5 min of synthesis, and the reaction mixture was incubated for 25 min at 35°C.…”

Stability of the cbb 3 -Type Cytochrome Oxidase Requires Specific CcoQ-CcoP Interactions

“…In vitro synthesis of cyt c 2 was performed using plasmid pC2P2.71, which carries the cycA gene under the control of both its own and the lac promoter. Cell-free protein synthesis using [ 35 S]methionine with R. capsulatus S-135 extracts was carried out for 30 min at 35°C as described previously (14,21,52). For cotranslational integration of in vitro-synthesized proteins into membranes, ICM were added after 5 min of synthesis, and the reaction mix was incubated for 25 min at 35°C.…”

In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways ofEscherichia coli

“…The purification of SecA (Helde et al, 1997), SecB (Hoffschulte et al, 1994), and F 1 -ATPase (Mü ller et al, 1987) followed previously reported protocols. His-tagged Ffh (P48) was expressed from pDS12-48His6 in E. coli strain MC4100 containing the lac repressor-encoding pDMI,1 (Lentzen et al, 1994) and purified in a manner similar to that described by Lentzen and associates.…”

“…Inside-out inner membrane vesicles (INV) and translocation factors were added from the beginning. Gradient-purified inner membrane vesicles were obtained as described previously (Hoffschulte et al, 1994) and extracted with 6 M urea as detailed elsewhere (Helde et al, 1997).…”