ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages.
The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.
Hou
Jie M
JM
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3125, USA.
D'Lima
Nadia G
NG
Rigel
Nathan W
NW
Gibbons
Henry S
HS
McCann
Jessica R
JR
Braunstein
Miriam
M
Teschke
Carolyn M
CM
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D015169:Colony Count, Microbial; D004795:Enzyme Stability; D008264:Macrophages / Q000382:microbiology*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000472:pathogenicity*; D011485:Protein Binding; D013696:Temperature; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D037521:Virulence Factors; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C442122:SecA1 protein, Mycobacterium tuberculosis; C442123:SecA2 protein, Mycobacterium