title: The location of ubiquitin in Lethocerus arthrin.
abstract: Arthrin is a ubiquitinated actin that is present in flight muscles of some insects. In addition, it has been found in the malaria parasite Plasmodium falciparum. The role of this monoubiquitylation is not clear, and it does not appear to be associated with proteolytic degradation. The stoichiometry of arthrin to actin in Lethocerus indirect flight muscle, 1:6, suggests that there would be one arthrin molecule for each Tm-Tn (tropomyosin-troponin) complex. The appearance of arthrin after tropomyosin and troponin in Drosophila development is consistent with the Tm-Tn complex determining which actin subunit is targeted for conjugation with ubiquitin. We have used a new approach of three-dimensional reconstruction of helical filaments, the iterative helical real space reconstruction method, to extract segments of homogeneous arthrin out of long filaments where the conformation of the ubiquitin is more heterogeneous. Surprisingly, the location of the ubiquitin is on the face of actin subdomain 1, opposite to where tropomyosin binds in the "off" state, suggesting that there could not be a direct interaction between the ubiquitin and the tropomyosin. It is possible that the troponin complex in the "on" state that is bound to one actin strand makes an unfavorable contact with a ubiquitin molecule attached to the opposite actin strand. This might be the basis for a destabilization of the on state at rest length. Lys118 is the most likely residue to which the ubiquitin is conjugated, based upon fitting atomic structures of actin and ubiquitin into the reconstruction.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Jordan Hill Box 800773, Charlottesville, VA 22908-0733, USA.
authors.forename: Vitold E
authors.initials: VE
authors.lastname: Galkin
authors.forename: Albina
authors.initials: A
authors.lastname: Orlova
authors.forename: Natalya
authors.initials: N
authors.lastname: Lukoyanova
authors.forename: Margaret S
authors.initials: MS
authors.lastname: VanLoock
authors.forename: Petra
authors.initials: P
authors.lastname: Hååg
authors.forename: Belinda
authors.initials: B
authors.lastname: Bullard
authors.forename: Edward H
authors.initials: EH
authors.lastname: Egelman
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D020062:Heteroptera / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D019476:Insect Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008958:Models, Molecular; D015394:Molecular Structure; D009124:Muscle Proteins / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D014335:Tropomyosin / Q000737:chemistry; D014336:Troponin / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D000199:Actins; D019476:Insect Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D009124:Muscle Proteins; D014335:Tropomyosin; D014336:Troponin; D025801:Ubiquitin; C045830:arthrin