MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination.
MURF-1, MURF-2 and MURF-3 are a specific class of RING finger proteins that are expressed in striated muscle tissues. MURF-1 has been suggested to act as an ubiquitin ligase, thereby controlling proteasome-dependent degradation of muscle proteins. Here, we performed yeast two-hybrid (YTH) screens of skeletal muscle cDNA libraries with MURF-1 baits to identify potential myocellular targets of MURF-1-dependent ubiquitination. This identified eight myofibrillar proteins as binding partners of MURF-1: titin, nebulin, the nebulin-related protein NRAP, troponin-I (TnI), troponin-T (TnT), myosin light chain 2 (MLC-2), myotilin and T-cap. YTH mating studies with MURF-1,2,3 baits indicated that these eight myofibrillar proteins are all targeted redundantly by both MURF-1 and MURF-2. Western blot studies on cardiac tissues from wild-type and MURF-1-deficient mice suggested that titin and nebulin were ubiquitinated at similar levels, and MLC-2 and TnI at reduced levels in MURF-1 KO mice. Mapping of the TnI and titin binding sites on MURF-1 peptide scans demonstrated their binding to motifs highly conserved between MURF-1 and MURF-2. Our data are consistent with a model in which MURF-1 and MURF-2 together target a specific set of myofibrillar proteins redundantly, most likely to control their ubiquitination-dependent degradation. Finally, our YTH screens identified the interaction of MURF-1 with 11 enzymes required for ATP/energy production in muscle including the mitochondrial ATP synthase and cytoplasmic creatine kinase. These data raise the possibility that MURF-1 may coordinately regulate the energy metabolism of mitochondrial and cytoplasmic compartments.
Witt
Stephanie H
SH
Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, Mannheim 68167, Germany.
Granzier
Henk
H
Witt
Christian C
CC
Labeit
Siegfried
S
D000595:Amino Acid Sequence; D000818:Animals; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D009210:Myofibrils / Q000378:metabolism*; D000070584:Tripartite Motif Proteins; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D009124:Muscle Proteins; D000070584:Tripartite Motif Proteins; D025801:Ubiquitin; C502755:muscle RING finger 2 protein, mouse; C440908:TRIM63 protein, human; C499084:Trim63 protein, mouse; D044767:Ubiquitin-Protein Ligases