purpose. To characterize the structure of cone βγ-transducin (Tβ3γ8) and its interaction with phosducin (pdc).

methods. The Tγ8 subunit of Tβ3γ8 was isolated by column chromatography for peptide mapping with mass spectrometry. Tβ3γ8 was compared with rod βγ-transducin (Tβ1γ1) in terms of the electrophoretic mobility, pdc binding affinity, and the effects of phosphorylation and methylation, and then the correlation to the crystal structures and functional domains of Tβ1γ1 was determined.

results. The mature Tγ8 is a 65-amino-acid peptide encoded by the Gγ8 gene with an acetylated and a farnesylated–methylated N-and C-terminus, respectively. Purified Tβ3γ8 is similar to Tβ1γ1 in that (1) both are heterogeneous, containing methylated and demethylated Tγ subunits;(2) each demethylated dimer migrates faster than its methylated counterpart during native gel electrophoresis, and the methylation-associated mobility differential is masked by pdc binding; and (3) both dimers bind pdc with the same affinity, and the affinity is reduced threefold by PKA phosphorylation of pdc and twofold by demethylation at the C-terminus of Tγ. Tβ3γ8 differs from Tβ1γ1 in exhibiting lower intrinsic electrophoretic mobility, and the difference is unaffected by either pdc binding or the status of Tγ methylation.

conclusions. Tβ3γ8 is identical with Tβ1γ1 in Tγ isoprenylation, the spatial organization, and the mode of pdc binding, indicating that its interaction with pdc does not play an important role in the specialization of cones. Changes in Tβγ characteristics by Tγ methylation reveal conformational changes on a surface domain that is essential for Tβγ functions and support a regulatory role for reversible methylation.