Poster Sessions

Biochem-2

Kristin Burns

K. Burns, H. Boshoff, C. Barry

The Search for Ubiquitin in Prokaryotes

Ubiquitin is a small, ubiquitous, highly conserved eukaryotic protein which is used to post-translationally modify cellular proteins to target them for degradation by the proteasome. The post-translational modification involves an isopeptide bond between the C-terminal glycine residue of ubiquitin and the -amino group of a lysine residue on the target protein. Ubiquitin is typically less than eighty amino acids, contains a C-terminal di-glycine motif, and has a unique-grasp fold. In addition to protein turnover, ubiquitin is involved in many cellular processes including protein regulation, trafficking and sorting. The proteasome-ubiquitin system was previously thought to exist only in eukaryotes until recent studies identified the proteasome in gram-positive actinobacteria. Reconstitution of the proteasome in several prokaryotes has shown that it can degrade short peptides, however, the true substrates have yet to be identified and a homolog of ubiquitin has not been found. The genes for the prokaryotic proteasome as well as putative accessory factors are clustered in the genome of these actinobacteria. The prokaryotic proteasome appears to be in an operon with a short open reading frame (ORF). This ORF encodes a small protein of about sixty-four amino acids with a di-glycine motif at the C-terminus. Although this protein shows little sequence homology to eukaryotic ubiquitin, its size, the di-glycine motif, as well as its proximity to the prokaryotic proteasome led us to believe this protein could be the prokaryotic version of ubiquitin. Indeed, when this protein was used as the bait in an in vivo pull-down assay in Mycobacterium smegmatis, several proteins appeared conjugated to it and this conjugation was dependent on the C-terminal residues. We are currently working to confirm the nature of the conjugation, and to test the conjugated proteins as substrates for the proteasome and its accessory factors in vitro. If confirmed as a substrate for the proteasome, this system could be an excellent tool to monitor protein turnover at various stages of cell growth. The presence of this system in Mycobacterium tuberculosis would also enable us to monitor its proteome in response to antibiotics and cellular insults.