Purchase this article with an account.
MS Obin, B Lee, C Thulin, A Bohm, RH Lee, BM Willardson, A Taylor; Phosphorylation- Dependent Binding of Phosducin (Pd) to Transducin(T) beta-gamma Blocks T gamma Ubiquitination Catalyzed by Ubiquitin (Ub)- Conjugating Enzyme (Ubc)H5 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1389.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:Using purified photoreceptors, we previously demonstrated that selective ubiquitination of Tßγ on Tγ targets Tßγ for proteolysis via the proteasome (Invest. Ophthalmol. Vis. Sci. 41(4), s607). In the present work, we identify regulators of Tßγ ubiquitination and Ub- dependent proteolysis . Methods:Tßγ and Pd-Tßγ complex were purified from bovine retina. Recombinant Pd was monophosphorylated (Pd∼P) with PKA and pentaphosphorylated (Pd∼P5) with CamIIK. Human Ubcs were expressed in bacteria. Tßγ complex were purified from bovine retina. Ubiquitination was assayed in retinal cell supernatants or fraction II from reticulocyte lysate (FII) and was detected by Western blot using anti- Tß and anti-Tγ IgGs. Results:Ubiquitination of Tßγ occured exclusively on Tγ, with preferential synthesis of mono-and tri-ubiquitinated species. Tßγ ubiquitination required FI or specific Ubcs present in FI (i.e, UbcH5 or UbcH7), but not Ubcs H1, H2, H3,H6 or H10. An active site mutant of UbcH5 inhibited Tßγ ubiquitination, suggesting that Tßγ is preferentially ubiquitinated via UbcH5. In contrast to native T ßγ, purified or in vitro -reconstituted Pd-Tßγ complex was entirely resistant to ubiquitination and degradation. However, Pd∼P5, which does not bind Tßγ, did not inhibit Tßγ ubiquitination; Pd∼P, which weakly binds Tßγ, partially inhibited Tßγ ubiquitination.Conclusion:Data suggest a novel mechanism of Tßγ regulation and quality control involving the Ub- proteasome pathway and light- regulated properties of Pd.
This PDF is available to Subscribers Only