Pup interacts with the ATPase Mpa and the proteasome substrate FabD. (A) Mpa interacted with Pup in an E. coli two-hybrid system. E. coli (cya) was transformed with combinations of plasmids encoding either of the two domains of Bordetella pertussis Cya, T25 (“plasmid 1”) or T18 (“plasmid 2”), fused to test proteins (for plasmid details, see fig. S1A and table S1). ‘Pup represents the 26–amino acid fragment identified from an Mtb genomic T25 library with T18C-Mpa as bait (a). Interactions that reconstituted functional Cya permitted growth on minimal lactose agar (“+”). All strains grew on minimal glucose agar (fig. S1A). (B) Mpa interacted with Pup in vitro. His₆-Pup, SigE-His₆, or E. coli “vector only” lysate on Ni-NTA agarose was incubated with recombinant Mpa (“input”). Fractions were separated by 15% SDS–polyacrylamide gel electrophoresis (PAGE) and visualized with Coomassie Brilliant Blue (CBB). The same samples were analyzed by anti-Mpa immunoblot (IB, below). (C) Pup interacted with FabD in an Msm two-hybrid system. Msm was transformed with combinations of plasmids encoding either of the two domains of murine dihydrofolate reductase, F(1,2) (“plasmid 1”) or F(3) (“plasmid 2”), fused to Pup, FabD, GCN4 (a Saccharomyces cerevisiae leucine zipper domain), or no other protein (for plasmid details, see fig. S1B and table S1). Positive interactions permitted growth on trimethoprim (Trim) (“+”). Pup had weak interactions with GCN4 (f, l). All strains grew on media lacking Trim (fig. S1B). (D) Pup formed a stable complex with FabD in Msm. FLAG-tagged proteins were enriched from equal amounts of lysates of Msm with plasmids encoding FLAG-FabD and either empty vector or His₆-Pup. Untagged FabD was the negative control. Samples were separated by 12% SDS-PAGE, and analyzed by anti-FLAG or anti-His₅ immunoblotting. FLAG-FabD migrated at the predicted size (arrow, left) and at a higher molecular size (fig. S3A); the ∼45-kD anti-His₅–reactive protein (asterisk, right) is only seen in mycobacteria producing FLAG-FabD and His₆-Pup.

Pupylation is associated with Mtb proteasome substrates. (A) Aberrant amounts of pupylation correlated with proteasome-defective states. Equal amounts of soluble Mtb lysates from WT, mpa and pafA strains were incubated with Ni-NTA agarose for enrichment of FLAG-FabD-His₆. Samples were deliberately overloaded to detect pupylated protein and observe the relative amounts of unpupylated versus pupylated FabD. Anti-FLAG immunoblots of Ni-NTA eluates detected both unpupylated (arrow) and pupylated (asterisk) FLAG-FabD-His₆. Anti-Pup immunoblots of the same samples detected Pup∼FLAG-FabD-His₆ (asterisk) in WT and mpa Mtb but not in the pafA strain. As a control, FLAG-DlaT-His₆ was purified from WT Mtb. Anti-FLAG immunoblots detected a protein at the predicted size of FLAG-DlaT-His₆, but no pupylated species was detected. Ponceau S staining shows that protein is present on this membrane (fig. S6B). (B) Multiple pupylated proteins were present in Mtb, but not in a pafA mutant. Anti-Pup immunoblots of Mtb lysates from WT, mpa, pafA, pafB, and pafC strains. Equivalent cell numbers were analyzed and the same blot was used for detection of endogenous DlaT. All samples were separated by 10% SDS-PAGE.

The C terminus of Mtb Pup covalently attaches to Lys¹⁷³ of Mtb FabD. (A) Alignment of the C terminus of Pup to that of Pup or ubiquitin from representative Actinomycetes or eukaryotes, respectively. Identical amino acids are shaded black. Sequences were compiled from the National Center for Biotechnology Information server and aligned by means of ClustalW (23). (B) Purification of the FabD∼Pup complex. Msm was cotransformed with plasmids encoding FLAG-FabD and His₆-Pup. FLAG-FabD∼His₆-Pup was purified sequentially with Ni-NTA agarose and anti-FLAG M2 affinity matrix. Proteins from each purification step were analyzed by 12% SDS-PAGE and visualized with CBB. (C) Tandem mass (MS/MS) spectrum of a FabD tryptic peptide derived by collision-induced dissociation of the (M + 2H)²⁺ precursor, mass/charge ratio (m/z) 869.963 [1.55 parts per million (ppm)]. Singly charged fragment ions marked in the spectrum represent peptide bond cleavage resulting in the sequence information recorded from both the N and C termini (b- and y-type ions, respectively). This spectrum, searched with the SEQUEST program, matched to the peptide shown with a mass shift corresponding to a deamidation event, converting the Pup C-terminal Gln to Glu (Q*). High mass accuracy MS/MS unambiguously confirms covalent modification of lysine in FLAG-FabD by His₆-Pup, with multiple matching b- and y-type ions. Additional detailed fragment ion information and additional spectra are presented in fig. S4. (D) Extracted ion chromatograms of the C-terminal peptide of Asp-N–digested His₆-Pup. The traces correspond to the m/z of MH₂²⁺ precursors ±3 ppm). In E. coli, deamidated Gln was detected at a low abundance (∼10%), whereas in Msm, the C-terminal Gln deamidation predominated. Q* denotes a deamidated Gln, equivalent to Glu. See fig. S5 and (10) for additional details. Amino acid residues: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.

Pupylation is required for Mpa-dependent protein degradation. (A) K173A mutation stabilized FabD. WT Msm expressing WT fabD or fabD with the K173 codon mutated to alanine was pulse labeled with ³⁵S-methionine and cysteine. Samples were collected over time and FLAG-FabD-His₆ (WT or K173A mutant) was purified and analyzed by 10% SDS-PAGE (10). This image represents a 12-hour exposure. A 6-hour exposure of the same gel is shown in fig. S7A. Immunoblot analysis showed that the K173A mutant was also not efficiently pupylated (fig. S7B). (B) Quantification of labeled protein in (A). (C) Pupylated proteins were degraded in an Mpa-dependent manner. WT and mpa mutant Msm were treated as in (A) and His₆-pupylated proteins were purified and analyzed. Total ³⁵S protein labeling is shown in fig. S7C. All data are representative of at least two independent experiments.