Abstract
Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6α, PDE6β, and two regulatory PDE6γ subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6α and PDE6β and can form a complex with prenyl-binding protein δ (PrBP/δ), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6–PrBP/δ binding and the mechanism by which the PDE6–PrBP/δ complex assembles have not been fully characterized, and the location of regulatory PDE6γ subunits within the protein assembly has not been elucidated. To clarify these questions, we have developed a rapid purification method for PDE6–PrBP/δ from bovine rod outer segments utilizing recombinant PrBP/δ. Transmission electron microscopy of negatively stained samples revealed the location of PrBP/δ and, thus, where the carboxyl-termini of PDE6α and PDE6β must be located. The three-dimensional structure of the PDE6αβγ complex was determined up to 18 Å resolution from single-particle projections and was interpreted by model building to identify the probable location of isoprenylation, PDE6γ subunits, and catalytic sites.
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