In vitro isoprenylation and membrane association of mouse rod photoreceptor cGMP phosphodiesterase alpha and beta subunits expressed in bacteria.
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.
Qin
N
N
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Pittler
S J
SJ
Baehr
W
W
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010786:Photoreceptor Cells / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D012717:Sesquiterpenes
D008565:Membrane Proteins; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C002963:geranylgeranyl pyrophosphate