The chaperone environment at the cytoplasmic face of the endoplasmic reticulum can modulate rhodopsin processing and inclusion formation.
The human DnaJ (Hsp40) proteins HSJ1a and HSJ1b are type II DnaJ proteins with different C termini generated by alternate splicing. Both protein isoforms can regulate the ATPase activity and substrate binding of Hsp70. In this study, we have confirmed the neuronal expression of HSJ1a and HSJ1b proteins and localized their expression in human neural retina using isoform-specific antisera. HSJ1a and HSJ1b were enriched in photoreceptors, particularly the inner segments, but had different intracellular localization due to the prenylation of HSJ1b by a geranylgeranyl 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 rhodopsin apoprotein in SK-N-SH cells. The expression of HSJ1b, but not HSJ1a, inhibited the normal cellular processing of wild-type rhodopsin-GFP, which co-localized with HSJ1b at the ER. 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 a prenylation-null mutant showed that the modulation of rhodopsin processing and inclusion formation was dependent on the correct subcellular targeting of HSJ1b to the cytosolic face of the ER. An Hsp70 interaction-null mutant of HSJ1b had the same effect as HSJ1b, suggesting that these phenomena were independent of Hsp70 and, furthermore, overexpression of Hsp70 with HSJ1b did not modulate the HSJ1b effect on inclusion formation, showing that Hsp70 was not limiting. The data provide evidence that cytoplasmic chaperones when targeted to the ER can influence the folding and processing of a GPCR and show that DnaJ protein function can be further specialized by alternative splicing and post-translational modification.
Chapple
J Paul
JP
Division of Pathology, Institute of Ophthalmology, University College of London, London EC1V 9EL, United Kingdom.
Cheetham
Michael E
ME
D000251:Adenosine Triphosphatases / Q000378:metabolism; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001923:Brain Chemistry; D002458:Cell Fractionation; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry*; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D006801:Humans; D007163:Immunosorbent Techniques; D002479:Inclusion Bodies; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010786:Photoreceptor Cells / Q000737:chemistry; D017510:Protein Folding; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000502:physiology; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D012160:Retina / Q000737:chemistry / Q000648:ultrastructure; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013116:Spinal Cord / Q000737:chemistry; D014018:Tissue Distribution; D014162:Transfection
C494647:DNAJB1 protein, human; C121958:DNAJB2 protein, human; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D020033:Protein Isoforms; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012243:Rhodopsin; D000251:Adenosine Triphosphatases