Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein

Abstract

The striking identification of an apparent proteasome core in Mycobacteria and allied actinomycetes suggested that additional elements of this otherwise strictly eukaryotic system for regulated protein degradation might be conserved. The genes encoding this prokaryotic proteasome are clustered in an operon with a short open reading frame that encodes a small protein of 64 amino acids resembling ubiquitin with a carboxyl-terminal di-glycine-glutamine motif (herein called Pup for prokaryotic ubiquitin-like protein). Expression of a polyhistidine-tagged Pup followed by pulldown revealed that a broad spectrum of proteins were post-translationally modified by Pup. Two-dimensional gel electrophoresis allowed us to conclusively identify two targets of this modification as myoinositol-1-phosphate synthase and superoxide dismutase. Deletion of the penultimate di-glycine motif or the terminal glutamine completely abrogated modification of cellular proteins with Pup. Further mass spectral analysis demonstrated that Pup was attached to a lysine residue on its target protein via the carboxyl-terminal glutamine with deamidation of this residue. Finally, we showed that cell lysates of wild type (but not a proteasome mutant) efficiently degraded Pup-modified proteins. These data therefore establish that, despite differences in both sequence and target linkage, Pup plays an analogous role to ubiquitin in targeting proteins to the proteasome for degradation.

FIGURE 1.

A, genomic organization of putative proteasome genes in M. smegmatis (Ms), Streptomyces coelicolor (Sc), and Rhodococcus erythropolis (Re). Proteasome-associated genes are in black; homologs are connected by dashed lines. Figure is adapted from Fig. 4 in Ref. . B, multalin alignment of Orf7 from various actinomycetes reveals a striking conservation in amino acid sequence at the carboxyl terminus.

FIGURE 2.

A, SDS-PAGE analysis of hisPup proteins eluted from Ni-NTA resin, detected with Coomassie Blue. Lane 1, hisPup; lane 2, hisPup purified under denaturing conditions; lane 3, Pup lacking his tag; lane 4, hisPupdGG, where the carboxyl-terminal GG motif is deleted; lane 5, hisPupdQ, where the carboxyl-terminal Gln is deleted (both hisPupdGG and hisPupdQ are denoted by an asterisk). B, anti-His Western analysis of SDS-PAGE in A (hisPupdGG and hisPupdQ do not transfer well and are revealed upon increased exposure in the inset). C, two-dimensional gel analysis of hisPup proteins eluted from Ni-NTA resin, detected with Coomassie Blue. Spot 1, superoxide dismutase; spot 2, superoxide dismutase and Pup; spot 3, myoinositol-1-phosphate synthase; spot 4, myoinositol-1-phosphate synthase and Pup.

FIGURE 3.

A, Ni-NTA purification of hisMips (^*) and its pupylated form (^**) following overexpression of both proteins in M. smegmatis. The third band of higher molecular weight was identified as GroEL by mass spectrometry. B, intact protein mass spectrometry analysis of hisMips (^*). C, intact protein mass spectrometry analysis of the pupylated hisMips (^**) protein. D, high resolution mass spectrometry data on the fragment containing the GGQ signature ion. E, tandem mass spectrometric data corresponding to the fragments of the ion observed in D.

FIGURE 4.

A, decay of pupylated proteins in wild type (left) and prcba mutant M. smegmatis (right) as observed by Western blotting with penta-His-horseradish peroxidase-conjugated antibody followed by chemiluminescence detection. B, quantification of A using ImageQuant (Amersham Biosciences). Wild type is shown in triangles; prcba mutant is shown in squares, and prcba mutant complemented with a vector containing prcba in diamonds and dashed line.