Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis.
Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and alpha adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.
Tam
B M
BM
Program in Neuroscience, Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA. btam@neuron.uchc.edu
Moritz
O L
OL
Hurd
L B
LB
Papermaster
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D049452:Green Fluorescent Proteins; D007814:Larva / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019814:Myristic Acid / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D011942:Receptors, Adrenergic, alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D017299:Rod Opsins / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D014982:Xenopus laevis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011942:Receptors, Adrenergic, alpha; D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D012243:Rhodopsin