title: Correlation of transducin photoaffinity labeling with the specific formation of intermolecular disulfide linkages in its α-subunit.
abstract: Transducin (T) is a heterotrimer of Tα, Tβ, and Tγ subunits. In the presence of light-activated rhodopsin, 8-azidoguanosine triphosphate (8-N3GTP) was covalently incorporated into T in a UV-light photodependent manner, with a low stoichiometry of 0.02 mol of 8-N3GTP per mol of T. Although Tα was preferentially labeled by 8-N3GTP, Tβ and Tγ were also modified. Photolabeling of T was specifically inhibited by GDP and GTP, but not by β,γ-imido-guanosine 5'-triphosphate (GMP-PNP), indicating that 8-N3GTP was modifying the GDP binding site of the holoenzyme. This was consistent with the observation that the photoaffinity probe was completely hydrolyzed to 8-N3GDP by T activated by illuminated rhodopsin. The formation of intermolecular disulfide associations in T was also determined because photolabeling of T was performed under non-reducing conditions. We established that Cys-347 of Tα was the major residue involved in the formation of disulfide-linked T oligomers. Other cysteines of Tα, such as Cys-321, also participated in the formation of disulfide bonds, revealing a complex pattern of intermolecular disulfide cross-links that led to the polymerization of T. The spontaneous generation of these cystines in Tα inhibited the light-dependent GTPase and GMP-PNP binding activities of T. A model was constructed illustrating that when two heterotrimers dimerize through the formation of disulfide bridges between the Cys-347 of their Tα subunits, the guanine ring of the 8-N3GDP bound to one T molecule might approach to the Tβγ-complex of the other heterotrimer. This model provides an explanation for the additional photolabeling of Tβ and Tγ by 8-N3GTP.
authors.affiliation: Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela. Electronic address: deisyperdomo@usb.ve.
authors.forename: Deisy
authors.initials: D
authors.lastname: Perdomo
authors.affiliation: Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela. Electronic address: carolina@rostonics.com.
authors.forename: Carolina
authors.initials: C
authors.lastname: Möller
authors.affiliation: Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela. Electronic address: jbubis@usb.ve.
authors.forename: José
authors.initials: J
authors.lastname: Bubis
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry; D002417:Cattle; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D008958:Models, Molecular; D019920:Photoaffinity Labels / Q000737:chemistry*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism
chemical_list: C021961:8-azidoguanosine triphosphate; D001386:Azides; D004220:Disulfides; D019920:Photoaffinity Labels; D021122:Protein Subunits; D006160:Guanosine Triphosphate; D015293:Transducin; D003545:Cysteine
keywords: Disulfide bonding; Oligomerization; Photoaffinity labeling; Transducin; Visual process