Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo.
Proteasome-bearing bacteria make use of a ubiquitin-like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C-terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Deltadop strain, pupylation is severely impaired and the steady-state levels of two known proteasomal substrates are drastically increased. Pupylation can be re-established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C-terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N-terminal ATP-binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.
Imkamp
Frank
F
ETH Zurich, Institute of Molecular Biology & Biophysics, Zurich, Switzerland.
Rosenberger
Tobias
T
Striebel
Frank
F
Keller
Peter M
PM
Amstutz
Beat
B
Sander
Peter
P
Weber-Ban
Eilika
E
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D025801:Ubiquitin / Q000378:metabolism
D001426:Bacterial Proteins; D025801:Ubiquitin; D005973:Glutamine; D046988:Proteasome Endopeptidase Complex; D000581:Amidohydrolases