MUF1/leucine-rich repeat containing 41 (LRRC41), a substrate of RhoBTB-dependent cullin 3 ubiquitin ligase complexes, is a predominantly nuclear dimeric protein.
RhoBTB (BTB stands for broad-complex, tramtrack, bric à brac) proteins are tumor suppressors involved in the formation of cullin 3 (Cul3)-dependent ubiquitin ligase complexes. However, no substrates of RhoBTB-Cul3 ubiquitin ligase complexes have been identified. We identified MUF1 (LRRC41, leucine-rich repeat containing 41) as a potential interaction partner of RhoBTB3 in a two-hybrid screening on a mouse brain cDNA library. MUF1 is a largely uncharacterized protein containing a leucine-rich repeat and, interestingly, a BC-box that serves as a linker in multicomponent, cullin 5 (Cul5)-based ubiquitin ligases. We confirmed the interaction of MUF1 with all three mammalian RhoBTB proteins using immunoprecipitation. We characterized MUF1 in terms of expression profile and subcellular localization, the latter also with respect to RhoBTB proteins. We found out that MUF1 is a ubiquitously expressed nuclear protein that, upon coexpression with RhoBTB, partially retains in the cytoplasm, where both proteins colocalize. We also show that MUF1 is able to dimerize similarly to other leucine-rich repeat-containing proteins. To explore the significance of MUF1-RhoBTB interaction within Cul-ligase complexes and the mechanism of MUF1 degradation, we performed a protein stability assay and found that MUF1 is degraded in the proteasome in a Cul5-independent manner by RhoBTB3-Cul3 ubiquitin ligase complex. Finally, we explored a possible heterodimerization of Cul3 and Cul5 and indeed discovered that these two cullins are capable of forming heterodimers. Thus, we have identified MUF1 as the first substrate for RhoBTB-Cul3 ubiquitin ligase complexes. Identification of substrates of these complexes will result in better understanding of the tumor suppressor function of RhoBTB.
Centre for Cardiovascular and Metabolic Research, The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK; Center for Biochemistry, Medical Faculty, and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany.
Kristína
K
Schenková
Centre for Cardiovascular and Metabolic Research, The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK.
Julia
J
Lutz
Center for Biochemistry, Medical Faculty, and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany.
Marion
M
Kopp
Center for Biochemistry, Medical Faculty, and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany.
Sonia
S
Ramos
Centre for Cardiovascular and Metabolic Research, The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK; Center for Biochemistry, Medical Faculty, and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany. Electronic address: francisco.rivero@hyms.ac.uk.
Francisco
F
Rivero
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D002522:Chlorocebus aethiops; D044842:Cullin Proteins / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008954:Models, Biological; D009687:Nuclear Proteins / Q000032:analysis*; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D059748:Proteolysis; D020798:Two-Hybrid System Techniques; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D002352:Carrier Proteins; C503646:Cul3 protein, mouse; D044842:Cullin Proteins; C576192:MUF1 protein, mouse; D009687:Nuclear Proteins; D020741:rho GTP-Binding Proteins