Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: characterization of TMs IV-VII and IX-XII and their accessibility to the aqueous translocation pathway.
We have determined the accessibility of the Rickettsia prowazekii ATP/ADP translocase transmembrane domains (TMs) IV-VII and IX-XII to the putative, water-filled ATP translocation pathway. A library of 177 independent mutants, each with a single cysteine substitution, was expressed in Escherichia coli, and those with substantial ATP transport activity were assayed for inhibition by thiol-reactive, methanethiosulfonate (MTS) reagents. The MTS reagents used were MTSES (negatively charged), MTSET (positively charged), and MTSEA (amphipathic). Inhibition of ATP transport by a charged MTS reagent indicates the exposure of a TM to the water-filled ATP translocation pathway. The eight TMs characterized in this study had 32 mutants with no assayable transport activity, indicating that cysteine substitution at these positions is not tolerated. ATP transport proficient mutants in TMs IV, V, VII, X, and XI were inhibited by charged MTS reagents, indicating that these TMs are exposed to the aqueous ATP translocation pathway, which is a pattern similar to those of TMs I, II (Alexeyev, M. F. (2004) Biochemistry 43, 6995-7002), and VIII (Winkler, H. H. (2003) Biochemistry 42, 12562-12569). Conversely, ATP-transport-proficient mutants in TMs VI, IX, and XII were not inhibited by charged MTS reagents, indicating that these TMs are sequestered from the aqueous environment, which is a pattern similar to that of TM III (Alexeyev, M. F. (2004) Biochemistry 43, 6995-7002). Preexposure of several MTS-sensitive mutants in TMs V, VII, X, and XI to ATP concentrations 10 times the K(m) resulted in protection from MTS-mediated inhibition; thus, confirming exposure of these TMs to the aqueous ATP translocation pathway, a pattern of protection similar to that observed for TMs I, II, and VIII.
Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama 36688, USA.
Jonathon P
JP
Audia
Rosemary A W
RA
Roberts
Herbert H
HH
Winkler
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics*; D008698:Mesylates / Q000378:metabolism / Q000494:pharmacology; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D012283:Rickettsia prowazekii / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D008698:Mesylates; D013438:Sulfhydryl Compounds; C074502:methanethiosulfonate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D003545:Cysteine