Isolation and Characterization of the Phage T4 PinA Protein, an Inhibitor of the ATP-dependent Lon Protease ofEscherichia coli

Jamese J Hilliard,Michael R Maurizi,Lee D Simon

doi:10.1074/jbc.273.1.518

Jamese J Hilliard, Michael R Maurizi + Show 1 more

Open Access

https://doi.org/10.1074/jbc.273.1.518

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Abstract

The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene. PinA is a heat-stable protein with a subunit Mr of 18,800 and an isoelectric point of 4.6. Under nondenaturing conditions on a gel filtration column, PinA migrated in two peaks corresponding to a dimer and a tetramer. Purified PinA inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E. coli ATP-dependent proteases, ClpAP or ClpYQ. Furthermore, PinA did not inhibit ATP-independent proteolysis in E. coli cell extracts. PinA binds with high affinity to Lon protease (Kd approximately 10 nM for dimer binding), and a complex with approximately 1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration. Lon activity was partially restored upon dilution of the PinA-Lon complex to subnanomolar concentrations, indicating that inhibition was reversible and that PinA did not covalently modify Lon protease. PinA was not cleaved by Lon protease, and heating the Lon-PinA complex at 65 degrees C denatured Lon protease and released active PinA. The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversible complex with Lon protease.

Highlights

The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene
Protein substrates bind to two sites on Lon, the proteolytic active site and an allosteric site, which may serve as the site for the protein remodeling function
E. coli cells infected with bacteriophage T4 show reduced proteolysis of abnormal proteins and protein fragments [18]

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals were obtained from commercial sources unless otherwise specified. [3H]Formaldehyde was obtained from NEN Life Science Products. After centrifugation for 15 min at 30,000 ϫ g, the pellet was suspended in 10 –15 ml buffer T with 0.1 M KCl. Insoluble matter was removed by centrifugation, and the resulting supernatant was loaded in aliquots of 2.5 ml onto separate 2.3 ϫ 60-cm TSK250 gel filtration columns equilibrated in buffer T with 0.1 M KCl. Proteins were eluted in the same buffer at a flow rate of 2 ml/min. Following cell lysis and centrifugation, the PinA protein was precipitated with ammonium sulfate as described above, and the pellet was suspended in PIPES buffer and dialyzed overnight. Proteins were eluted from the Superose-12 column in 20 mM PIPES, pH 6.0, containing 0.5 M NaCl, 1 mM ␤-mercaptoethanol, and 1 mM EDTA buffer, at a flow rate of 0.2 ml/min. Lon (250 ␮g), PinA (250 ␮g), and mixtures of the two proteins were analyzed by gel filtration on a Superose column equilibrated in buffer B. To determine if formation of the PinA-Lon complex was reversible, the complex isolated by gel filtration was diluted into assay solutions of increasing volume (100 ␮l to 1.6 ml) to reduce the concentrations of PinA and Lon below the apparent Kd, and the increase in Lon activity was measured

RESULTS

Fresh Lon treated with heated eluates

DISCUSSION

Mixture of PinA and Lon

Methods