The overall mechanism of the Cdc complex resembles
The overall mechanism of the Cdc48 complex resembles that of the 19S regulatory subunit of the proteasome, which also uses receptor proteins to bind polyubiquitin chains attached to a substrate and employs a translocation mechanism (for review, see Kish-Trier and Hill, 2013). As with Cdc48, full substrate movement through the central pore requires deubiquitination, a reaction performed by Rpn11 of the 19S subunit. In (-)-p-Bromotetramisole Oxalate to Otu1, which cleaves between ubiquitin moieties and does not fully remove a substrate-associated ubiquitin chain, Rpn11 cleaves off the entire chain. However, in both cases, deubiquitination cooperates with, rather than antagonizes, substrate processing by the ATPase complex. This is distinct from the function of other DUBs, such as the Ubp6 component of the 19S subunit, which serve as negative regulators.
In many cellular functions, such as ERAD, Cdc48 acts upstream of the proteasome. Cdc48 may be required in cases where a substrate does not have a flexible segment (Beskow et al., 2009), which is needed to initiate translocation into the 19S proteasomal subunit (Prakash et al., 2004). Indeed, in our experiments, the Cdc48 complex can translocate a polyubiquitin chain, which does not expose extended flexible segments. The exact reason why Cdc48, but not the proteasome, can deal with folded substrates remains to be clarified. The mechanism of substrate transfer from Cdc48 to the proteasome also needs further investigation. Most of the ubiquitin chains released by Cdc48 would be long enough to bind directly, or through the shuttling factors Rad23 or Dsk2, to the proteasome (Kim et al., 2004, Medicherla et al., 2004), and chains that are too short could first be extended by the Cdc48-associated “E4 ligase” Ufd2 (Richly et al., 2005). Alternatively, some substrates may be transferred from Cdc48 into the 20S proteasome without the involvement of the 19S subunit (Barthelme and Sauer, 2012, Barthelme and Sauer, 2013). Regardless of the downstream events, our results show that Cdc48 pulls on substrate polypeptides and unfolds them, explaining how the ATPase complex disassembles protein complexes and extracts proteins from membranes.
Acknowledgments We thank Alex Stein for providing reagents; the Institute for Chemistry and Chemical Biology Longwood for use of a plate reader; and Dan Finley, Alex Stein, and Ryan Baldridge for critical reading of the manuscript. This work is supported by the NIH/NIGMS Award R01GM052586 and by award #T32GM007753 from the NIGMS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. T.A.R. is a Howard Hughes Medical Institute Investigator.
Introduction Mycobacterium tuberculosis (Mtb), causes more than 10 million new cases of tuberculosis and about 1.8 million deaths . Latent tuberculosis infection occurs when the pathogen enters a dormant state and persists in the human body without causing active clinical disease . ATP binding cassette (ABC) proteins play critical roles in all organisms . The ABC-F subfamily of ABC proteins contain tandem nucleotide-binding domains (NBDs) but no transmembrane domains (TMDs). Unlike the membrane-localized ATP transporters, these soluble proteins are involved in cellular processes such as translational control and antibiotic resistance by ribosomal protection , . The Mtb genome encodes several ABC family proteins. Although several TMD-containing ABC transporters in Mtb have been shown to be involved in transport processes including antibiotic efflux, the soluble Mtb ABC proteins that contain tandem NBDs but lack TMDs have not been studied , . The Rv2477c protein encoded in the Mtb genome shows strong sequence identity with EttA, the ABC-F protein in Escherichia coli, that associates with the ribosome to control translation . The transcript levels of Rv2477c were upregulated when the pathogen was subjected to antibiotic stress . Recently, it was reported that a multi-drug resistant Mtb strain isolated from a human patient harbored a single nucleotide polymorphism in the gene encoding Rv2477c that altered the pathogen's phenotype from antibiotic susceptible to resistant . In spite of these indications of its potential importance, Rv2477c has not been studied. Therefore, we investigated the biochemical characteristics of Rv2477c in this study.