Characterization of the autophosphorylation of Era, an essential Escherichia coli GTPase.
Era is an essential protein in Escherichia coli which binds both GTP and GDP and has an intrinsic GTPase activity. Studies on the role of GTP/GDP binding and GTPase activity in an attempt to understand its function lead to the observation that Era is autophosphorylated. The autophosphorylation reaction is specific for GTP and cannot use ATP as a phosphoryl group donor. The reaction velocity is of first order with respect to protein concentration, suggesting an intramolecular mechanism. Autophosphorylation occurs at serine and threonine residues. The major phosphorylated tryptic peptide isolated after autophosphorylation has been identified as ISITSR, from residue 33 to 38. The peptide contains the site of phosphorylation and two potential sites for serine and threonine phosphorylation. Subsequently, both the threonine residue at position 36 and the serine residue at position 37 were altered to alanine. The double mutant Era, but not individual single mutants, was unable to functionally complement the growth of an E. coli strain which cannot produce wild-type Era protein at high temperature. This suggests that either threonine 36 or serine 37 has to exist for the function of Era in vivo. In vivo phosphorylation of Era was also examined by two-dimensional gel electrophoresis. Era has been previously assigned two distinct positions having two different X-Y co-ordinates: one of the spots (H032.0) was identified as phosphorylated Era, indicating that a substantial portion of Era in the cell is indeed phosphorylated. Therefore, Era autophosphorylation is likely to play an important physiological role in the cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Sood
P
P
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School at Rutgers, Piscataway 08854.
Lerner
C G
CG
Shimamoto
T
T
Lu
Q
Q
Inouye
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; C068027:era protein, E coli; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins