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Characterization of the protein composition of detergent‐resistant microdomains and detergent‐soluble fractions from retinal rod outer segment membranes prepared under various conditions
Author(s) -
Bubis Jose,
Perdomo Deisy Belen
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a614-d
Subject(s) - gtp' , phosphorylation , immunoprecipitation , rhodopsin , transducin , western blot , lipid raft , microbiology and biotechnology , membrane , chemistry , biochemistry , biology , biophysics , retinal , enzyme , gene
Detergent‐resistant membrane microdomains (DRMs) were isolated from bovine rod outer segments (ROS) under dark and light conditions, and in the absence or presence of ATP, GTPγS or both. ROS‐derived DRMs were enriched in caveolin‐1 (Cav) and c‐Src. Although the 50‐kDa form of arrestin was mainly present in the illuminated ROS‐derived detergent soluble fractions (DSFs), the 44‐kDa truncated form (p44) was localized in both, DRMs and DSFs. Under all conditions tested, rhodopsin (R) was present in DRMs and DSFs. No localization difference was seen in the presence of ATP for any of the above proteins. In the dark, transducin (T) was unphosphorylated and mainly present in the DSF, but following illumination it was Tyr‐phosphorylated and mobilized toward the DRM fraction. In the light, GTPγS released T into the DSF; however, T was retained in the DRMs when ATP was added following treatment with GTPγS. These results suggest that T phosphorylation prevents its GTPγS‐induced release from the DRMs. Moreover, immunoprecipitation assays followed by Western blot analyses under light conditions clearly showed the formation of multiprotein complexes containing R, T, c‐Src, p44 and Cav in DRMs, in which T and c‐Src were Tyr‐phosphorylated. The association of these proteins to the DRMs appears to be mediated by an anchoring or scaffolding protein, which could be Cav. Supported by a grant from FONACIT N° S1‐2514.