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Ameair Abu Irqeba, Judy Ogilvie; COPI Coated Vesicles and PRA1 Co-localize and are Mis-localized in Differentiating rd1 Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3738. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The rd1 mouse is a well-studied model of Retinitis Pigmentosa (RP), a disease that leads to the degeneration of photoreceptors. Beyond the premature stop codon in the mutant Pde6b gene, the events that promote the degeneration phenotype are not clearly understood. The gene encoding the Prenylated Rab Acceptor 1 (PRA1) protein is consistently down-regulated prior to the onset of degeneration in the rd1 retina. Studies have proposed that PRA1 plays a role in the trafficking of Rab GTPases. It is unknown if this protein's function is conserved in photoreceptors and what effect its down-regulation has in the in the rd1 retina.
To gain insight into PRA1 function, we used the split-ubiquitin yeast-two hybrid system to find novel interactors. Interactions were confirmed in Hek293 cells using the bimolecular fluorescence complementation (BiFC) method. Co-localization was demonstrated using immunohistochemistry combined with the expression of a PRA1-GFP construct (under the control of the CAG promoter), injected subretinally into wild type retina and electroporated at P0 and harvested at P21.
We observed that the PRA1 protein is prominently expressed in photoreceptors and is prevalent in the inner segment, does not get trafficked to the outer segment, and co-localizes with the ribbon synapse marker RIBEYE. Among the interacting proteins found using the split-ubiquitin yeast two hybrid system was the COPI coat subunit Copz1, whose interaction was confirmed in Hek293 cells. In the adult wild type photoreceptors, COPI vesicles are prevalent in the synapse, inner segment and outer segment and co-localize with RIBEYE as well as PRA1-GFP constructs. Staining at P10 (prior to the onset of photoreceptor degeneration in the rd1 retina) shows that COPI coated vesicles are enriched in the photoreceptor synapse in the wild type retina, but accumulate at the inner segment in rd1 photoreceptors.
COPI vesicular trafficking to the synapse during the maturation process appears to be hindered in rd1 photoreceptors, which may lead to erroneous trafficking to organelles in the inner segment. Hindrance of trafficking to the synapse is consistent with the down-regulation of PRA1 in the rd1 retina.
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