The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation.

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with DeltaF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.

[1]  T. Dryja,et al.  Transgenic mice with a rhodopsin mutation (Pro23His): A mouse model of autosomal dominant retinitis pigmentosa , 1992, Neuron.

[2]  J. O'Tousa.  Requirement of N-linked glycosylation site in Drosophila rhodopsin , 1992, Visual Neuroscience.

[3]  I. Wada,et al.  A novel ER α‐mannosidase‐like protein accelerates ER‐associated degradation , 2001 .

[4]  P. Sieving,et al.  Retinopathy induced in mice by targeted disruption of the rhodopsin gene , 1997, Nature Genetics.

[5]  N. Colley,et al.  Role of Asparagine-linked Oligosaccharides in Rhodopsin Maturation and Association with Its Molecular Chaperone, NinaA* , 2000, The Journal of Biological Chemistry.

[6]  J. Nathans,et al.  Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[7]  E. L. Kean,et al.  Effect of enzymatic deglycosylation on the regenerability of bovine rhodopsin. , 1992, Experimental eye research.

[8]  Junying Yuan,et al.  Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-β , 2000, Nature.

[9]  P. Sieving,et al.  P23H rhodopsin transgenic rat: correlation of retinal function with histopathology. , 2000, Investigative ophthalmology & visual science.

[10]  H. Lehrach,et al.  Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. , 2001, Molecular biology of the cell.

[11]  O. L. Moritz,et al.  A Functional Rhodopsin-Green Fluorescent Protein Fusion Protein Localizes Correctly in Transgenic Xenopus laevis Retinal Rods and Is Expressed in a Time-dependent Pattern* , 2001, The Journal of Biological Chemistry.

[12]  David W. Yandell,et al.  A point mutation of the rhodopsin gene in one form of retinitis pigmentosa , 1990, Nature.

[13]  C J Lumsden,et al.  Inherited neurodegenerative diseases: the one-hit model of neurodegeneration. , 2001, Human molecular genetics.

[14]  I. Wada,et al.  A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation. , 2001, EMBO reports.

[15]  E. Sztul,et al.  Characterization and Dynamics of Aggresome Formation by a Cytosolic Gfp-Chimera✪ , 1999, The Journal of cell biology.

[16]  C. Zuker,et al.  Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[17]  C. Maggioni,et al.  Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[18]  S. Kaushal,et al.  Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa. , 1994, Biochemistry.

[19]  R. Plemper,et al.  Retrograde protein translocation: ERADication of secretory proteins in health and disease. , 1999, Trends in biochemical sciences.

[20]  F. Tokunaga,et al.  N‐Linked glycosylation of Drosophila rhodopsin occurs exclusively in the amino‐terminal domain and functions in rhodopsin maturation , 1998, FEBS letters.

[21]  J. Chillarón,et al.  Dissociation from BiP and retrotranslocation of unassembled immunoglobulin light chains are tightly coupled to proteasome activity. , 2000, Molecular biology of the cell.

[22]  S. Basinger,et al.  Tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[23]  A. Helenius,et al.  Intracellular functions of N-linked glycans. , 2001, Science.

[24]  S. Jentsch,et al.  Role of the proteasome in membrane extraction of a short‐lived ER‐transmembrane protein , 1998, The EMBO journal.

[25]  J. Nathans,et al.  Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[26]  R. Kopito,et al.  Impairment of the ubiquitin-proteasome system by protein aggregation. , 2001, Science.

[27]  A. Goldberg,et al.  Cellular Defenses against Unfolded Proteins A Cell Biologist Thinks about Neurodegenerative Diseases , 2001, Neuron.

[28]  R. Kopito,et al.  Aggresomes: A Cellular Response to Misfolded Proteins , 1998, The Journal of cell biology.

[29]  A. Milam,et al.  Rhodopsin transgenic pigs as a model for human retinitis pigmentosa. , 1998, Investigative ophthalmology & visual science.

[30]  M. Cheetham,et al.  Unfolding retinal dystrophies: a role for molecular chaperones? , 2001, Trends in molecular medicine.

[31]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2000, Science.

[32]  U. Wolfrum,et al.  Rhodopsin’s Carboxy-Terminal Cytoplasmic Tail Acts as a Membrane Receptor for Cytoplasmic Dynein by Binding to the Dynein Light Chain Tctex-1 , 1999, Cell.

[33]  T. Wileman,et al.  Aggresomes Resemble Sites Specialized for Virus Assembly , 2001, The Journal of cell biology.

[34]  H. Khorana,et al.  Structure and function in rhodopsin. Requirements of a specific structure for the intradiscal domain. , 1994, The Journal of biological chemistry.

[35]  B. Rosner,et al.  Effect of vitamin A supplementation on rhodopsin mutants threonine-17 --> methionine and proline-347 --> serine in transgenic mice and in cell cultures. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[36]  K. Willison,et al.  Mutations in the N-terminus of the X-linked retinitis pigmentosa protein RP2 interfere with the normal targeting of the protein to the plasma membrane. , 2000, Human molecular genetics.

[37]  C. M. Davenport,et al.  Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain. , 1993, The Journal of biological chemistry.

[38]  S. Warren,et al.  Aberrant Protein Deposition and Neurological Disease* , 1999, The Journal of Biological Chemistry.

[39]  D. Baylor,et al.  A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[40]  M. Naash,et al.  Opsin localization and rhodopsin photochemistry in a transgenic mouse model of retinitis pigmentosa , 1998, Neuroscience.

[41]  Rahul S. Rajan,et al.  Specificity in intracellular protein aggregation and inclusion body formation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[42]  R. E. Kalina,et al.  Clinical-ultrastructural study of a retinal dystrophy. , 1983, Investigative ophthalmology & visual science.

[43]  M. Figueiredo-Pereira,et al.  Ubiquitin, cellular inclusions and their role in neurodegeneration , 1998, Trends in Neurosciences.

[44]  P. Kurada,et al.  Retinal degeneration caused by dominant rhodopsin mutations in Drosophila , 1995, Neuron.

[45]  S. Kaushal,et al.  Structure and function in rhodopsin: the role of asparagine-linked glycosylation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[46]  O. L. Moritz,et al.  Identification of an Outer Segment Targeting Signal in the Cooh Terminus of Rhodopsin Using Transgenic Xenopus laevis , 2000, The Journal of cell biology.

[47]  Satoshi Omura,et al.  Degradation of CFTR by the ubiquitin-proteasome pathway , 1995, Cell.

[48]  M. Gurney,et al.  Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[49]  R. Kopito,et al.  Aggresomes, inclusion bodies and protein aggregation. , 2000, Trends in cell biology.

[50]  M. Adamian,et al.  Rhodopsin accumulation at abnormal sites in retinas of mice with a human P23H rhodopsin transgene. , 1994, Investigative ophthalmology & visual science.

[51]  H. Ploegh,et al.  Intracellular targeting of the proteasome. , 2000, Trends in cell biology.