ClpXP-dependent proteolysis of FNR upon loss of its O2-sensing [4Fe-4S] cluster.
The global regulator FNR from Escherichia coli controls the transcription of genes required for an anaerobic lifestyle. While previous studies have demonstrated that FNR activity is regulated by O2 through loss of dimerization upon destruction of its [4Fe-4S]2+ cluster, the present study reveals that monomeric FNR protein is also a target of proteolysis. We have found that turnover of FNR protein is increased selectively under aerobic growth conditions, when FNR is not active as a transcription factor and is primarily a metal-free, monomeric form (apo-FNR). This degradation of monomeric FNR was dependent on the ClpXP protease and required the presence of two amino acid sequences within FNR that resemble known ClpX recognition motifs. By measuring the turnover rates of various FNR mutants that have unique properties with respect to dimerization and Fe-S cluster stability, we have shown that loss of dimerization upon [4Fe-4S]2+ cluster destruction by O2 targets FNR for degradation by the ClpXP protease. In addition, by measuring the differential rate of FNR degradation upon switching aerobic cultures to anaerobic growth conditions, we provide evidence that pre-existing FNR apo-protein can be converted to [4Fe-4S]2+ -FNR. Finally, we address the physiological significance of FNR proteolysis by demonstrating that varying FNR protein levels over a small range under aerobic growth conditions has a direct effect on the function of FNR in O2 sensing.
Mettert
Erin L
EL
Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53706, USA.
Kiley
Patricia J
PJ
D000332:Aerobiosis; D000693:Anaerobiosis; D015374:Biosensing Techniques; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D049071:Endopeptidase Clp / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000032:analysis* / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D001616:beta-Galactosidase / Q000378:metabolism
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D001616:beta-Galactosidase; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D010100:Oxygen