PrtD, the integral membrane ATP-binding cassette component of the Erwinia chrysanthemi metalloprotease secretion system, exhibits a secretion signal-regulated ATPase activity.
We have overproduced, partially purified, and characterized PrtD, the ATP-binding cassette (ABC) integral membrane component from the metalloproteases secretion system of the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi. These metalloproteases are secreted independently of the general export pathway encoded by the sec genes. They are secreted via a C-terminal secretion signal and by a secretion apparatus composed of two inner membrane proteins, PrtD and PrtE, and one outer membrane protein PrtF. PrtD is specifically labeled by 8-azido-ATP both in whole membrane vesicles and upon purification. The purified protein displays a low level of P-type ATPase activity. This activity is almost completely and specifically inhibited by the cognate C-terminal secretion signal of the PrtG and PrtB metalloproteases (half inhibition at 0.1 microM) but not by a C-terminal secretion signal of a protein not secreted by the Prt translocator. A mutant PrtD protein bearing a point mutation in the ATP binding site (conserved lysine 370 of the Walker A box changed to arginine) has also been purified. It displays a lower level of ATPase activity which correlates with the lower level of secretion of the metalloproteases by a strain expressing this mutated protein.
Unité de Physiologie Cellulaire, Institut Pasteur (Centre National de la Recherche Scientifique, URA 1300), Paris, France.
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D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D016974:Dickeya chrysanthemi / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping
D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D001426:Bacterial Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; C090391:PrtD protein, Erwinia chrysanthemi; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008666:Metalloendopeptidases; D000251:Adenosine Triphosphatases