May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
The Chaperone Environment at the Cytoplasmic Face of the Endoplasmic Reticulum Can Modulate Rhodopsin Processing and Inclusion Formation
Author Affiliations & Notes
  • J.P. Chapple
    Pathology, Inst Ophthalmology-UCL, London, United Kingdom
  • M.E. Cheetham
    Pathology, Inst Ophthalmology-UCL, London, United Kingdom
  • Footnotes
    Commercial Relationships  J.P. Chapple, None; M.E. Cheetham, None.
  • Footnotes
    Support  BBSRC, EC, MRC and Wellcome Trust
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2845. doi:
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      J.P. Chapple, M.E. Cheetham; The Chaperone Environment at the Cytoplasmic Face of the Endoplasmic Reticulum Can Modulate Rhodopsin Processing and Inclusion Formation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2845.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Modulating rhodopsin folding and degradation through manipulation of the cell chaperone machinery could have important implications for developing therapies for RP caused by misfolding mutations in rhodopsin. The human DnaJ (Hsp40) proteins HSJ1a and HSJ1b have different C-termini generated by alternate splicing from a single copy gene. They are preferentially expressed in neuronal tissues and can regulate the substrate binding of Hsp70. We have investigated the localisation of HSJ1a and HSJ1b in neural retina and examined the effect of these chaperones on the processing of rhodopsin. Methods: Peptide directed antisera to HSJ1a and HSJ1b were used to determine the localisation of the HSJ1 isoforms in human retina, by immunocytochemistry. Intracellular targeting of HSJ1b was investigated by mutagenesis within a putative prenylation motif. Co-localisation between HSJ1 isoforms and rhodopsin-GFP was examined by confocal microscopy and interactions between the proteins resolved by co-immunoprecipitation. The effect of HSJ1 on rhodopsin processing was further analysed by the quantification of inclusion formation. Results: HSJ1a and HSJ1b were enriched in photoreceptors, particularly the inner segments, but had different intracellular localization due to the prenylation of HSJ1b by a gernaylgeranyl moiety. Because of their enrichment at the site of rhodopsin production, we investigated the effect of HSJ1 isoforms on the cellular processing of wild type and mutant P23H rhodopsin apoprotein in SK-N-SH cells. The expression of HSJ1b, but not HSJ1a, inhibited the normal cellular processing of wild type rhodopsin-GFP, rhodopsin-GFP was retained in the ER and co-localized with HSJ1b. HSJ1b expression also increased the incidence of inclusion formation by the wild type protein. Both isoforms were recruited to mutant P23H rhodopsin inclusions, but only HSJ1b enhanced inclusion formation. Investigation of H31Q and C321S mutants of HSJ1b suggested that the modulation of rhodopsin processing and inclusion formation was independent of the interaction with Hsp70, but dependent on the correct sub-cellular targeting of HSJ1b to the cytosolic face of the ER. Conclusions: These data provide evidence that cytoplasmic chaperones can influence the folding and processing of rhodopsin. Understanding the specialized chaperone networks within photoreceptors will be essential to exploit the potential of cellular chaperone machines to manipulate the folding of normal and mutant rhodopsin.

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