A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium.
The HemA enzyme (glutamyl-tRNA reductase) catalyzes the first committed step in heme biosynthesis in the enteric bacteria. HemA is mainly regulated by conditional protein stability; it is stable and, consequently, more abundant in heme-limited cells but unstable and less abundant in normally growing cells. Both the Lon and ClpAP energy-dependent proteases contribute to HemA turnover in vivo. Here we report that the addition of two positively charged lysine residues to the third and fourth positions at the HemA N terminus resulted in complete stabilization of the protein. By contrast, the addition of an N-terminal myc epitope tag did not affect turnover. This result confirms the importance of the N-terminal sequence for proteolysis of HemA. This region of the protein also contains a proline flanked by hydrophobic residues, a motif that has been suggested to be important for Lon-mediated proteolysis of UmuD. However, mutation of this motif did not affect the turnover of HemA protein. Cells expressing the stabilized HemA[KK] mutant protein display substantial defects in heme regulation.
Wang
L
L
Department of Microbiology and Immunology, West Virginia University Health Sciences Center, Morgantown, West Virginia 26506, USA.
Wilson
S
S
Elliott
T
T
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D049071:Endopeptidase Clp; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005246:Feedback; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006360:Heat-Shock Proteins / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D049070:Protease La; D012352:RNA, Transfer, Glu / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D012352:RNA, Transfer, Glu; D011994:Recombinant Proteins; D006418:Heme; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D008239:Lysine