Coordination between production and turnover of interphotoreceptor retinoid-binding protein in zebrafish.
OBJECTIVE
Interphotoreceptor retinoid-binding protein (IRBP), which is secreted by the photoreceptors of most vertebrates, is the major soluble protein component of the interphotoreceptor matrix (IPM). Recent studies suggest that IRBP is short lived in the IPM (half-life, approximately 11 hours). The mechanisms coordinating the production and removal of IRBP are not known. Zebrafish provide a useful system to study the regulation of these two processes, because its IRBP mRNA levels are under circadian regulation. In the present study, the relationship between the quantity of IRBP, the rate of its turnover, and the expression of its mRNA in the zebrafish retina were examined.


METHODS
Full-length zebrafish IRBP was expressed in Escherichia coli and an antiserum generated against purified recombinant IRBP. Western and protein dot blot analyses and indirect immunofluorescence were used to define the temporal and spatial patterns of IRBP expression in the adult zebrafish. In vivo and in vitro metabolic labeling experiments were used to examine the regulation of IRBP turnover by both environmental light and the light-dark cycle.


RESULTS
Despite the known rhythmicity in IRBP mRNA expression, neither the amount of IRBP nor its localization changes significantly during the light-dark cycle. IRBP is rapidly removed from the zebrafish eye (half life, approximately 7 hours). This rapid turnover is independent of environmental lighting conditions during subjective day and is more rapid during the day than at night.


CONCLUSIONS
Because the amount of IRBP remains constant throughout the day, the enhanced daytime IRBP mRNA expression may function to compensate for an increased turnover of the protein during the day. These findings suggest that the processes of IRBP production and removal are coordinately regulated.
Cunningham
L L
LL
Graduate Program in Neuroscience, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Gonzalez-Fernandez
F
F
D000818:Animals; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D003623:Dark Adaptation; D004926:Escherichia coli / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006207:Half-Life; D015151:Immunoblotting; D008027:Light; D012333:RNA, Messenger / Q000096:biosynthesis; D012160:Retina / Q000378:metabolism*; D012177:Retinol-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015027:Zebrafish / Q000378:metabolism*; D000099214:Retinol-Binding Proteins, Interstitial
D017931:DNA Primers; D005136:Eye Proteins; D012333:RNA, Messenger; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial