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bioRxiv · 10.1101/2022.05.30.494045

Dual Palmitoylation of PRCD, a Photoreceptor-Specific Protein Linked to RP, Alters Protein Stability and Subcellular Localization

Myers, B. O. R.·Sechrest, E.·Hamner, G.·Motipally, S.·Murphy, J.·Kolandaivelu, S.
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Abstract

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (POS) disc-specific protein essential for maintaining outer segment (OS) structures, while also contributing to rhodopsin packaging densities and distribution in the disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation is found linked with retinitis pigmentosa (RP) that is crucial for protein stability and trafficking to POS. PRCD has several predicted structural domains with unknown significance, such as the polybasic region (PBR) where an Arg17Cys (R17C) mutation is linked with RP. In this study, we demonstrate that a mutation in the PBR augments additional palmitoyl lipid modification observed through acyl-RAC in the mutant cysteine (R17C). Immunolocalization of transiently expressed R17C protein in hRPE1 cells depicts similar characteristics to wild type (WT); however, a double mutant lacking endogenous palmitoylation at the Cys2 position is comparable to the C2Y protein as both are likely aggregated and mislocalized in the mitochondria. Subretinal injection of C2Y, R17C, and R17C/C2Y mutants followed by electroporation in murine retina exhibit mislocalization in the inner segment compared to WT PRCD. Our results in the R17C mutant show palmitoylation transpires at two different locations. Despite being dually palmitoylated and demonstrating membrane association, the mutation in the PBR affects protein stability and trafficking to the OS. Moreover, palmitoylation within the PBR alone does not compensate for protein stability or trafficking, revealing the PBR domain is indispensable and any defects likely lead to dysregulation of PRCD protein associated with blinding diseases.

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Myers, B. O. R., Sechrest, E., Hamner, G., Motipally, S., Murphy, J., Kolandaivelu, S.. 2022-05-30. Dual Palmitoylation of PRCD, a Photoreceptor-Specific Protein Linked to RP, Alters Protein Stability and Subcellular Localization. https://doi.org/10.1101/2022.05.30.494045

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