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Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.
Pubmed ID: 28943312 RIS Download
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Commercial antibody vendor which supplies antibodies and other products to life science researchers.
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View all literature mentionsCell line HeLa is a Cancer cell line with a species of origin Homo sapiens
View all literature mentionsCell line HEK293 is a Transformed cell line with a species of origin Homo sapiens (Human)
View all literature mentionsThis monoclonal targets phospho-Histone H2A.X (Ser139) clone JBW301 biotin conjugate
View all literature mentionsThis unknown targets Rabbit IgG (H+L)
View all literature mentionsThis monoclonal targets Ubiquitinylated proteins
View all literature mentionsThis monoclonal targets Mfn2
View all literature mentionsThis monoclonal targets Ubiquitin, Lys63-Specific
View all literature mentionsThis monoclonal targets Ubiquitin
View all literature mentionsThis polyclonal targets Mouse IgG (H+L)
View all literature mentionsThis unknown targets IgG
View all literature mentionsThis monoclonal secondary targets IgG
View all literature mentionsThis unknown targets Streptavidin-HRP
View all literature mentionsThis polyclonal targets IKBKG
View all literature mentionsThis polyclonal targets Tom20 (FL-145)
View all literature mentionsThis monoclonal targets Ubiquitin, Lys48-Specific
View all literature mentionsThis polyclonal targets Lasu1/Ureb1
View all literature mentionsThis monoclonal targets HA
View all literature mentionsThis monoclonal targets actin
View all literature mentionsThis unknown targets Rabbit IgG (H+L)
View all literature mentionsThis polyclonal targets IKBKG
View all literature mentionsThis monoclonal targets Ubiquitinylated proteins
View all literature mentionsThis monoclonal targets Mfn2
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View all literature mentionsThis monoclonal targets Ubiquitin, Lys63-Specific
View all literature mentionsThis monoclonal targets Ubiquitin
View all literature mentionsThis polyclonal targets Mouse IgG (H+L)
View all literature mentionsThis unknown targets IgG
View all literature mentionsThis monoclonal secondary targets IgG
View all literature mentionsThis unknown targets Streptavidin-HRP
View all literature mentionsThis polyclonal targets Tom20 (FL-145)
View all literature mentionsThis monoclonal targets Ubiquitin, Lys48-Specific
View all literature mentionsThis polyclonal targets Lasu1/Ureb1
View all literature mentionsThis monoclonal targets HA
View all literature mentionsThis monoclonal targets actin
View all literature mentionsThis monoclonal targets phospho-Histone H2A.X (Ser139) clone JBW301 biotin conjugate
View all literature mentionsThis monoclonal targets phospho-Histone H2A.X (Ser139) clone JBW301 biotin conjugate
View all literature mentionsThis monoclonal targets phospho-Histone H2A.X (Ser139) clone JBW301 biotin conjugate
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