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Abstract
Cytochrome b562 is not cleaved by the tail-specific protease Tsp in vitro or in the periplasm of Escherichia coli but becomes a good substrate when the C-terminal sequence WVAAA is added. Following randomization of the final three residue positions of this substrate, 54 different mutants with single residue substitutions were recovered. The steady-state expression levels of cytochrome variants bearing these mutant tails were similar in an E. coli strain deleted for the tsp gene but differed markedly in a strain containing Tsp. Wild-type cytochrome b562 and seven variants, displaying a range of intracellular expression levels, were purified. These proteins were found to have the same Tm values in thermal denaturation experiments but to be cleaved by Tsp at rates differing by as much as 30-fold. Overall, the rates of Tsp cleavage of these proteins in vitro correlate with their rates of cleavage in vivo as determined by pulse-chase experiments. These results indicate that the C-terminal sequence of the cytochrome-b562 variants is important in determining their proteolytic fate in the cell and show that this degradation is mediated predominantly by Tsp. There are different selectivity rules at each of the three C-terminal positions. The identity of the C-terminal residue of the substrate, where small, uncharged residues (Ala, Cys, Ser, Thr, Val) are preferred, is most important in determining the rate of substrate cleavage by Tsp. Non-polar residues are also preferred at the second and third positions, but larger and more hydrophobic side chains are also acceptable at these positions in good substrates.
Highlights
The observed selectivity of intracellular proteolysis implies the existence of mechanisms that allow proteases to discriminate between correct and incorrect protein substrates [1, 2]
Specific degradation of proteins with non-polar C-terminal sequences was first reported for several cytoplasmic proteins in Escherichia coli [4, 5], but the protease that mediates this degradation has not been identified
We show that wild-type cytochrome b562 is resistant to Tsp-mediated cleavage but becomes a good substrate when a WVAAA C-terminal tail is added
Summary
Plasmids, and Mutagenesis—E. coli strain X90 is ara ⌬(lac pro) gyrA argE(Am) RifϪ thi-1/FЈ lacIq lacϩ proϩ; E. coli strain KS1000 is X90 ⌬tsp(prc)3::kan eda-51::Tn10 [8]. The cell pellet was resuspended in 10 ml of chloroform, incubated at room temperature for 15 min, and 100 ml of 10 mM Tris-HCl (pH 8.0) was added This mixture was centrifuged for 20 min at 4,000 rpm, and the red aqueous supernatant containing cytochrome b562 and other periplasmic proteins was recovered. Pulse-Chase Assays—The half-lives of cytochrome-b562 variants in vivo were determined by growing cells to mid-log phase in M9 minimal medium containing no Cys or Met and inducing protein expression by addition of 1 mM isopropyl-1-thio--D-galactopyranoside. Cleavage Assays—The cleavage of cytochrome-b562 variants by Tsp in vitro was monitored by the change in heme absorbance, which accompanies its dissociation from the cleaved protein For this assay, 2.5 M of the purified cytochrome-b562 variant was incubated at 22 °C with 0.3 M Tsp-His in 50 mM Tris-HCl (pH 8.0), 20 mM NaCl, and the loss of absorbance at 418 nm was monitored Melting curves were fit to a two-state transition between native and denatured protein by non-linear least squares fitting using the program NONLIN for Macintosh [13, 14]
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