Mycobacterium tuberculosis Proteasome Accessory Factor A (PafA) Can Transfer Prokaryotic Ubiquitin-Like Protein (Pup) between Substrates.

Susan Zhang,Huilin Li,K Heran Darwin,Lizbeth Hedstrom,Kristin E Burns-Huang,Huib Ovaa,Guido V Janssen,Eric J Rubin

doi:10.1128/mbio.00122-17

Abstract

ABSTRACTThe protein degradation machinery of Mycobacterium tuberculosis includes a proteasome and a ubiquitin-like protein (Pup). Proteasome accessory factor A (PafA) attaches Pup to proteins to target them for degradation by the proteasome. Free Pup is unstable and never observed in extracts of M. tuberculosis, an observation that led us to hypothesize that PafA may need alternative sources of Pup. Here, we show that PafA can move Pup from one proteasome substrate, inositol 1-phosphate synthetase (Ino1), to two different proteins, malonyl coenzyme A (CoA)-acyl carrier protein transacylase (FabD) and lonely guy (Log). This apparent “transpupylation” reaction required a previously unrecognized depupylase activity in PafA, and, surprisingly, this depupylase activity was much more efficient than the activity of the dedicated depupylase Dop (deamidase of Pup). Thus, PafA can potentially use both newly synthesized Pup and recycled Pup to doom proteins for degradation.

Highlights

The protein degradation machinery of Mycobacterium tuberculosis includes a proteasome and a ubiquitin-like protein (Pup)
We found that Proteasome accessory factor A (PafA), unlike Dop, could not deamidate PupGln to PupGlu; PafA amidase activity appears to be limited to pupylated proteins
We used untagged M. smegmatis Dop copurified with M. tuberculosis Pup lacking the first 30 amino acids and with a His6 epitope tag (His6-Pup91); coproduction of His6-Pup91 allowed the production of soluble and active Dop

Summary

Introduction

The protein degradation machinery of Mycobacterium tuberculosis includes a proteasome and a ubiquitin-like protein (Pup). Pup is attached to target proteins via the gamma carboxylate (␥carboxylate) of the C-terminal glutamate residue In some species, such as Mycobacterium tuberculosis, the C-terminal amino acid is glutamine (PupGln), and the enzyme Dop (deamidase of Pup) must convert this glutamine to glutamate [3]. This step is required in order to allow the Pup ligase, PafA, to use ATP to phosphorylate (in contrast to adenylate for Ub) the ␥-carboxylate of the terminal glutamate. The ␧-amino group of a lysine in a doomed protein can attack the phosphoglutamate to form an isopeptide bond [4] (Fig. 1A) This reaction is similar to glutamine synthesis, which is a condensation reaction between glutamate and ammonia. In addition to deamidating PupGln to PupGlu, Dop can remove Pup from proteins, which can rescue them from proteasomal degradation [6, 8, 9]

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Results

Conclusion