The cGMP phosphodiesterase-transducin complex of retinal rods. Membrane binding and subunits interactions.
cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is composed of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Native PDE alpha beta gamma 2 is peripherally bound to the membranes of ROS discs. We studied quantitatively its partition between soluble and membrane-bound fractions in ROS homogenates. In the presence of its activator, the alpha-subunit of transducin loaded with a triphosphate guanine nucleotide (T alpha*), PDE displayed a greatly enhanced membrane binding. Neither the purified PDE gamma.T alpha* complex, nor the PDE alpha beta and PDE alpha beta gamma forms of active PDE, showed a membrane binding comparable to that of PDE alpha beta gamma 2 in the presence of T alpha*. The T alpha*-activated PDE is therefore an undissociated complex tightly bound to the ROS membranes. Using limited proteolysis, we showed that the membrane anchoring of the whole complex implies not only PDE (mainly by the C terminus of PDE beta) but also both termini of T alpha*. The membrane binding of the purified PDE alpha beta species was also enhanced in the presence of T alpha*; a direct link would therefore exist between the activator and the catalytic subunits. From this work emerges a plausible structural model of the T alpha*-activated PDE, with its internal interactions and its sites of anchoring into the ROS membrane.
Catty
P
P
Laboratoire de Biophysique Moléculaire et Cellulaire, Unité Associée 520 du Centre National de la Recherche Scientifici, Centre d'Etudes Nucléaires, Grenoble, France.
Pfister
C
C
Bruckert
F
F
Deterre
P
P
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002451:Cell Compartmentation; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D046911:Macromolecular Substances; D010786:Photoreceptor Cells / Q000378:metabolism*; D011485:Protein Binding; D015293:Transducin / Q000378:metabolism*
D046911:Macromolecular Substances; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin