ISOLATION AND CHARACTERIZATION OF THE PHAGE-T4 PINA PROTEIN INHIBITOROF THE ATP-DEPENDENT LON PROTEASE OF ESCHERICHIA-COLI

The bacteriophage T4 PinA protein, expression of which leads to inhibi tion of protein degradation in Escherichia coil cells, has been purifi ed from cells carrying multiple copies of the pinA gene. PinA is a hea t-stable protein with a subunit M-r of 18,800 and an isoelectric point of 4.6, Under nondenaturing conditions on a gel filtration column, Pi nA migrated in two peaks corresponding to a dimer and a tetramer, Puri fied Pink inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E. coli ATP-depende nt proteases, ClpAP or ClpYQ, Furthermore, PinA did not inhibit ATP-in dependent proteolysis in E. coil cell extracts, PinA binds with high a ffinity to Lon protease (K-d similar to 10 nM for dimer binding), and a complex with similar to 1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration, Lon activity was partially resto red upon dilution of the PinA-Lon complex to subnanomolar concentratio ns, indicating that inhibition was reversible and that PinA did not co valently modify Lon protease, PinA was not cleaved by Lon protease, an d heating the Lon-PinA complex at 65 degrees C denatured Lon protease and released active Pink The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversib le complex with Lon protease.