In vitro isoprenylation and membrane association of mouse rod photoreceptor cGMP phosphodiesterase alpha and beta subunits expressed in bacteria.

Academic Article

Abstract

  • We investigated the specificity of CAAX box-related isoprenylation of rod photoreceptor cGMP phosphodiesterase (PDE) subunits expressed in bacteria and the consequences of this modification on rod disk membrane association. Full-length cDNA sequences of the alpha and beta subunits of mouse PDE, inserted into bacterial pET expression vectors, were overexpressed as fusion proteins containing 28 (bMP-alpha) and 26 (bMP-beta) additional amino acid residues at their N termini. Both fusion proteins were overexpressed and stored in inclusion bodies. Purified bMP-alpha and bMP-beta were recognized by bovine PDE-specific polyclonal antibodies, but did not associate with depleted rod disk membranes and were catalytically inactive. Using bovine brain or retina extracts as sources of protein prenyltransferases and tritiated farnesyl- or geranylgeranylpyrophosphate as donors, bMP-alpha (CAAX sequence CCIQ) was exclusively farnesylated, and bMP-beta (CAAX sequence CCIL) was exclusively geranylgeranylated. After isoprenylation, bMP-alpha and bMP-beta each associated with rod photoreceptor outer segment disk membranes under isotonic, but not under hypotonic, conditions. The results indicate that isoprenylated bMP-alpha and bMP-beta each interact independently with membranes and that isoprenylation is the key modification that facilitates membrane association.
  • Authors

    Published In

    Keywords

  • 3',5'-Cyclic-GMP Phosphodiesterases, Amino Acid Sequence, Animals, Base Sequence, Blotting, Western, Cattle, Cell Membrane, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Gene Expression, Membrane Proteins, Mice, Molecular Sequence Data, Photoreceptor Cells, Polyisoprenyl Phosphates, Rod Cell Outer Segment, Sesquiterpenes
  • Author List

  • Qin N; Pittler SJ; Baehr W
  • Start Page

  • 8458
  • End Page

  • 8463
  • Volume

  • 267
  • Issue

  • 12