Structural studies of arthrin: monoubiquitinated actin.
Here, we report on the structure and in situ location of arthrin (monoubiquitinated actin). Labelling of insect muscle thin filaments with a ubiquitin antibody reveals that every seventh subunit along the filament long-pitch helices is ubiquitinated. A three-dimensional reconstruction of frozen-hydrated arthrin filaments was produced. This was based on a novel algorithm that divides filament images into short segments that are used for single-particle image processing. Difference maps with an actin filament reconstruction locate ubiquitin at the side of actin sub-domain 1 opposite where myosin binds. Consistent with the reconstructions, peptide mapping places the ubiquitin linkage on lysine 118 in actin. Molecular modelling was used to generate arthrin monomers from ubiquitin and actin crystal structures. Filament models constructed from these monomers were compared with the arthrin reconstruction. The reconstruction suggests ubiquitin attached to Lys118 adopts one or a few conformers, stabilized by a small interface with actin. The function of actin ubiquitination is not known, but may involve regulation of muscle contractile activity.
School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK.
Stan
S
Burgess
Matt
M
Walker
Peter J
PJ
Knight
John
J
Sparrow
Stephan
S
Schmitz
Gerald
G
Offer
Belinda
B
Bullard
Kevin
K
Leonard
John
J
Holt
John
J
Trinick
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D025801:Ubiquitin / Q000378:metabolism*
D000199:Actins; D029721:Drosophila Proteins; D019476:Insect Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D025801:Ubiquitin; C045830:arthrin