Abstract
Purpose :
Progressive Rod Cone Degeneration (PRCD) is a 54 amino acid long palmitoylated protein that resides in the disc membrane of the photoreceptor outer segments. Six mutations in PRCD, including C2Y, R17C, R18X, R22X, P25T, and V30M, have been linked to the development of retinitis pigmentosa in humans. Our goal was to determine the effects of each of these mutations on the expression and subcellular localization of PRCD.
Methods :
Constructs encoding wild type or mutant epitope-tagged human PRCD, in frame with IRES-GFP, were transiently expressed in human RPE1 cultured cells. The expression and stability of PRCD in the cells was monitored using Western blotting; its compartmentalization was determined by immunocytochemistry and subcellular membrane fractionation. The palmitoylation of PRCD was assayed using acyl resin-assisted capturing.
Results :
We found the C2Y and the R17C mutants to be 95% and 30% less stable, respectively, in the cells than the wild type PRCD. The V30M and the P25T mutations had no significant effect on the PRCD stability. The palmitoylation status of all the mutants, except for C2Y, remained unaltered. Furthermore, the levels of all mutants, except C2Y, and of wild type PRCD could be up-regulated by co-transfecting the cells with the zDHHC3 enzyme, which catalyzes protein palmitoylation. The immuno-staining of wild type PRCD was punctate and present throughout the cytoplasm. Most interestingly, the C2Y mutant predominantly accumulated in the mitochondria, while the R17C mutant was present in both in the mitochondria and in the cytoplasm, and the V30M mutant was retained in the Golgi. The compartmentalization of the P25T mutant was essentially the same as that of the wild type PRCD.
Conclusions :
Our results support the notion that these mutations of PRCD have specific effects on its posttranslational lipid modification, subcellular targeting, and ER-Golgi transport, all of which may potentially affect its normal function in retinal photoreceptors.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.