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© The Rockefeller University Press, 0021-9525/2000//1369 $5.00
The Journal of Cell Biology, Volume 151, Number 7, , 2000 1369-1380

Identification of an Outer Segment Targeting Signal in the Cooh Terminus of Rhodopsin Using Transgenic Xenopus laevis

^a Program in Neuroscience, Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030
Dept. of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030-6125.(860) 679-3693(860) 679-2020

btam{at}neuron.uchc.edu

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 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.

Key Words: rhodopsin • photoreceptors • transgenic animals • membrane proteins • cell polarity

© 2000 The Rockefeller University Press

Abbreviations used in this paper: AAR, 2A adrenergic receptor; dpf, days postfertilization; mGFP, myristoylated GFP; RIS, rod inner segment; ROS, rod outer segment; TR-WGA, Texas red–conjugated wheat germ agglutinin.

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