Identification of functionally important acidic residues in transducin by group-specific labeling

Abstract

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the α-subunit (Tα,) and the βγ-complex (Tβγ). The role of the carboxyl groups in T was evaluated by labeling with N, N′-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of β, γ-imido guanosine 5′-triphosphate (GMPpNp to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when Tα was combined with DCCD-treated Tβγ. However, the binding of guanine nucleotides to the reconstituted T was ∼50% inhibited when DCCD-labeled Tα was incubated with Tβγ. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified Tα was combined with Tβγ, and when EDC-treated Tβγ was incubated with Tα.