Prokaryotic ubiquitin-like protein provides a two-part degron to Mycobacterium proteasome substrates

Abstract

Prokaryotic ubiquitin-like protein (Pup) is a posttranslational modifier that targets proteins for degradation by the mycobacterial proteasome. We show that the disordered amino terminus of Pup is required for degradation, while the helical carboxyl terminus mediates its attachment to proteins. Thus, Pup has distinct regions that either interact with pupylation enzymes or initiate proteasomal degradation.

FIG. 1.

Fusion of Pup to a nonproteasome substrate targets it for proteasomal degradation in an Mpa- and proteasome-dependent manner. Representations of linear fusions expressed in M. tuberculosis (Mtb) and M. smegmatis (Msm) are shown above each panel. (A) M. tuberculosis H37Rv Zur-His₆ (Rv2359) is similarly stable at steady state in all M. tuberculosis and M. smegmatis strains tested. (B) Pup-Zur-His₆ accumulates in an mpa mutant but not in wild-type or pafA M. tuberculosis strains (left). Pup-Zur-His₆ accumulates in ΔprcBA but not wild-type M. smegmatis. The entire pup gene was fused to zur. The terminal Pup Gln codon was changed to Glu. In three independent experiments, less fusion protein was detected in the pafA mutant than in wild-type M. tuberculosis. (C) Deletion of nucleotides 4 to 90 (Δ91; deletion of amino acids 2 to 30) or 92 to 195 (N-term; deletion of amino acids 31 to 64) resulted in fusion genes that were equally stable in wild-type and degradation-defective strains (right). Experiments are representative of two independent replicate samples for each strain. Dihydrolipoamide acyltransferase (DlaT) is the loading control. Mutants are described elsewhere (1, 6). All cultures were processed as described elsewhere (7). For immunoblot analysis, cell lysates were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed with monoclonal antibodies to His₅ (Qiagen) or polyclonal antibodies to DlaT (a gift from R. Bryk and C. Nathan).

FIG. 2.

The Pup C terminus is sufficient for pupylation. Ectopic expression of his₆-pup or his₆-pup91 (ΔN-term-Pup) and subsequent Ni-nitrilotriacetic acid (NTA) purification of pupylated proteins from M. smegmatis. (A) Coomassie brilliant blue (CBB)-stained SDS-PAGE gel of Ni-NTA purified pupylomes. (B) Immunoblot (IB) analysis of purified proteins using antibodies to His₅. (C) Anti-Ino1 immunoblot shows Ino1 is pupylated with either full-length or truncated Pup. The pupylomes were purified from wild-type M. smegmatis cultures harvested at an optical density at 580 nm (OD₅₈₀) of 1. The amount of protein loaded was normalized based on protein concentration of the eluted proteins, as determined by A₂₈₀ using a NanoDrop spectrophotometer.

FIG. 3.

Expression of pup91 abrogates Ino1 degradation. Ectopic expression of his₆-pup91 abrogates degradation of endogenous Ino1. Equivalent cell numbers were harvested, lysed by bead beating, and clarified by microcentrifugation for 10 min at 16,000 × g. Antibodies to Ino1 are described elsewhere (9). DlaT is the loading control.