Comparative characterization of SecA from the alpha-subclass purple bacterium Rhodobacter capsulatus and Escherichia coli reveals differences in membrane and precursor specificity.
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.
Helde
R
R
Adolf Butenandt Institut für Physikalische Biochemie, Ludwig-Maximilians-Universität München, Munich, Germany.
Wiesler
B
B
Wachter
E
E
Neubüser
A
A
Hoffschulte
H K
HK
Hengelage
T
T
Schimz
K L
KL
Stuart
R A
RA
Müller
M
M
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003574:Cytochrome c Group / Q000096:biosynthesis; D045362:Cytochromes c2; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; D045362:Cytochromes c2; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins