Delineation of the plasma membrane targeting domain of the X-linked retinitis pigmentosa protein RP2.
OBJECTIVE
The X-linked retinitis pigmentosa protein RP2 is predominantly targeted to the plasma membrane. This study delineates the exact amino acid sequence requirements for targeting of RP2 through dual N-terminal acyl modification.


METHODS
Inhibition of acyl modification with a palmitate analogue was used to confirm the mechanism of intracellular targeting. Mutagenesis of the first 15 residues in a synthetic RP2-green fluorescent protein (GFP) chimera was used to probe the precise requirements for plasma membrane targeting in Chinese hamster ovary (CHO) cells by confocal microscopy and subcellular fractionation.


RESULTS
The N-terminal Met-Gly-Cys-X-Phe-Ser-Lys motif of human RP2 is necessary and sufficient for the protein's plasma membrane localization. This motif includes the accepted consensus sequence for N-myristoyl transferase (NMT) and a site for attachment of a palmitoyl moiety. An interesting feature of the motif is an essential phenylalanine at position 5. This is the first report of the requirement of a specific residue at position 5 within the N-terminal acyl modification motif for normal intracellular targeting. Arginine at position 8 is not essential for plasma membrane localization of the protein, but it improves targeting. The motif is highly conserved and is found in all vertebrate orthologues of human RP2, except mouse. In mouse, however, the Ser6Thr change is concordant with the accepted NMT consensus sequence.


CONCLUSIONS
Conserved residues mediate the intracellular targeting of RP2, further highlighting the potential significance of the protein's plasma membrane localization. The delineation of this motif identifies residues in which mutations disrupt the dual acylation of RP2 and almost certainly result in disease.
Chapple
J Paul
JP
Department of Pathology, Institute of Ophthalmology, University College London, London, United Kingdom.
Hardcastle
Alison J
AJ
Grayson
Celene
C
Willison
Keith R
KR
Cheetham
Michael E
ME
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D005136:Eye Proteins; D019204:GTP-Binding Proteins; D008040:Genetic Linkage; D049452:Green Fluorescent Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012174:Retinitis Pigmentosa / Q000235:genetics; D016415:Sequence Alignment; D014960:X Chromosome / Q000235:genetics
D005136:Eye Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011506:Proteins; C072178:RP2 protein, human; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins