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• W2000429226 abstract "Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes. Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes. outer segment extracellular/intradiskal loop 2 monoclonal antibody wild-type retinal degeneration slow insertional mutant The outer segments (OSs)1 of rod and cone photoreceptor cells act as detectors of visible light for the initial stages of the visual process and are vital for normal vertebrate vision. The OS is an architecturally complex organelle. It contains hundreds of precisely stacked membranous disks that undergo a polarized renewal process; a complete turnover of OS membrane protein occurs approximately once every 10 days in primate rod cells (1Young R.W. Invest. Ophthalmol. Vis. Sci. 1976; 15: 700-725PubMed Google Scholar). Although the renewal process and OS stability are essential for photoreceptor viability, their underlying molecular bases remain largely undefined. Evidence from several laboratories has implicated the integral membrane protein peripherin/rds in OS morphogenesis and the renewal process (2Molday R.S. Hicks D. Molday L. Invest. Ophthalmol. Vis. Sci. 1987; 28: 50-61PubMed Google Scholar, 3Travis G.H. Brennan M.B. Danielson P.E. Kozak C.A. Sutcliffe J.G. Nature. 1989; 338: 70-73Crossref PubMed Scopus (314) Google Scholar, 4Sanyal S. Jansen H.G. Neurosci. Lett. 1981; 21: 23-26Crossref PubMed Scopus (166) Google Scholar). It is present in all vertebrate rod and cone photoreceptors examined to date and causes a variety of progressive retinal diseases in humans when defective (5Kohl S. Giddings I. Besch D. Apfelstedt-Sylla E. Zrenner E. Wissinger B. Acta Anat. (Basel). 1998; 162: 75-84Crossref PubMed Google Scholar). Despite continued interest and study, neither the normal molecular action of peripherin/rds in OS renewal nor its role in the pathophysiology of retinal disease is well understood. Several suggestions for molecular function have been proposed based largely on the molecular genetic identification of peripherin/rds as the primary trigger for the murine retinal degeneration slow (rds) phenotype and the immunochemical localization of this protein to rod and cone OS disc rim regions (6Arikawa K. Molday L.L. Molday R.S. Williams D.S. J. Cell Biol. 1992; 116: 659-667Crossref PubMed Scopus (231) Google Scholar, 7Travis G.H. Groshan K.R. Lloyd M. Bok D. Neuron. 1992; 9: 113-119Abstract Full Text PDF PubMed Scopus (109) Google Scholar). Proposals for function include stabilization of the highly curved disc rims, maintenance of flattened disc structure through adhesive interactions, morphogenesis of disc membranes/rim regions, catalysis of disc shedding, and/or scaffolding of disc stacks (2Molday R.S. Hicks D. Molday L. Invest. Ophthalmol. Vis. Sci. 1987; 28: 50-61PubMed Google Scholar, 8Travis G.H. Sutcliffe J.G. Bok D. Neuron. 1991; 6: 61-70Abstract Full Text PDF PubMed Scopus (246) Google Scholar, 9Molday R.S. Invest. Ophthalmol. Vis. Sci. 1998; 39: 2491-2513PubMed Google Scholar, 10Goldberg A.F. Molday R.S. Biochemistry. 1996; 35: 6144-6149Crossref PubMed Scopus (107) Google Scholar, 11Boesze-Battaglia K. Lamba O.P. Napoli Jr., A.A. Sinha S. Guo Y. Biochemistry. 1998; 37: 9477-9487Crossref PubMed Scopus (73) Google Scholar). The recent finding that OS morphogenesis as well as photoreceptor function can be rescued in postnatal (10-day-old) rds mice reinforces the notion that this protein is a building block for the normally continuous OS renewal process (12Ali R.R. Sarra G.M. Stephens C. Alwis M.D. Bainbridge J.W. Munro P.M. Fauser S. Reichel M.B. Kinnon C. Hunt D.M. Bhattacharya S.S. Thrasher A.J. Nat. Genet. 2000; 25: 306-310Crossref PubMed Scopus (297) Google Scholar). In addition, the reported flattening of canine pancreatic microsomes by peripherin/rds in vitro is consistent with a direct role in OS structure (13Wrigley J.D. Ahmed T. Nevett C.L. Findlay J.B. J. Biol. Chem. 2000; 275: 13191-13194Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Current studies are directed at resolving the molecular details of peripherin/rds action. The cloning and identification of nearly a dozen highly conserved peripherin/rds orthologs (from humans to fish) has suggested that the proteins share several distinctive features, including four hydrophobic transmembrane segments, a large extracellular/intradiskal loop domain (EC2), seven conserved cysteine residues, and a covalently attached carbohydrate moiety (3Travis G.H. Brennan M.B. Danielson P.E. Kozak C.A. Sutcliffe J.G. Nature. 1989; 338: 70-73Crossref PubMed Scopus (314) Google Scholar, 14Travis G.H. Christerson L. Danielson P.E. Klisak I. Sparkes R.S. Hahn L.B. Dryja T.P. Sutcliffe J.G. Genomics. 1991; 10: 733-739Crossref PubMed Scopus (129) Google Scholar, 15Moghrabi W.N. Kedzierski W. Travis G.H. Exp. Eye Res. 1995; 61: 641-643Crossref PubMed Scopus (10) Google Scholar, 16Kedzierski W. Moghrabi W.N. Allen A.C. Jablonski-Stiemke M.M. Azarian S.M. Bok D. Travis G.H. J. Cell Sci. 1996; 109: 2551-2560Crossref PubMed Google Scholar, 17Weng J. Belecky-Adams T. Adler R. Travis G.H. Invest. Ophthalmol. Vis. Sci. 1998; 39: 440-443PubMed Google Scholar, 18Connell G.J. Molday R.S. Biochemistry. 1990; 29: 4691-4698Crossref PubMed Scopus (141) Google Scholar, 19Bascom R.A. Manara S. Collins L. Molday R.S. Kalnins V.I. McInnes R.R. Neuron. 1992; 8: 1171-1184Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 20Gorin M.B. Snyder S. To A. Narfstrom K. Curtis R. Mamm. Genome. 1993; 4: 544-548Crossref PubMed Scopus (15) Google Scholar, 21Moritz O.L. Molday R.S. Invest. Ophthalmol. Vis. Sci. 1996; 37: 352-362PubMed Google Scholar, 22Gould D.J. Petersen-Jones S.M. Lin C.T. Sargan D.R. Anim. Genet. 1997; 28: 391-396Crossref PubMed Scopus (14) Google Scholar). Hydrodynamic studies performed under reducing conditions indicate that WT peripherin/rds polypeptides can self-assemble to form homotetramers and also can co-assemble with rom-1 (a homologous polypeptide) to form heterotetramers (10Goldberg A.F. Molday R.S. Biochemistry. 1996; 35: 6144-6149Crossref PubMed Scopus (107) Google Scholar, 23Goldberg A.F. Moritz O.L. Molday R.S. Biochemistry. 1995; 34: 14213-14219Crossref PubMed Scopus (97) Google Scholar). The noncovalently associated tetramers can join via disulfide bonds to generate larger polymers of indeterminate size; polymerization appears to be mediated by a single conserved cysteine residue in peripherin/rds and inhibited by the presence of rom-1 (24Goldberg A.F. Loewen C.J. Molday R.S. Biochemistry. 1998; 37: 680-685Crossref PubMed Scopus (102) Google Scholar, 25Loewen C.J. Molday R.S. J. Biol. Chem. 2000; 275: 5370-5378Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Although tetramerization and polymerization are believed integral for function, their place in the mechanism of peripherin/rds action has yet to be established. Because OS formation is observed in the complete absence of rom-1 (26Clarke G. Goldberg A.F. Vidgen D. Collins L. Ploder L. Schwarz L. Molday L.L. Rossant J. Szel A. Molday R.S. Birch D.G. McInnes R.R. Nat. Genet. 2000; 25: 67-73Crossref PubMed Scopus (131) Google Scholar), peripherin/rds-containing tetramers appear to be the essential units of function for OS morphogenesis. Several instances of inherited retinal degeneration have been associated with the disruption of peripherin/rds folding and subunit assembly, yet other cases do not appear to impact these processes at all (24Goldberg A.F. Loewen C.J. Molday R.S. Biochemistry. 1998; 37: 680-685Crossref PubMed Scopus (102) Google Scholar, 27Goldberg A.F. Molday R.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13726-13730Crossref PubMed Scopus (105) Google Scholar). Such observations of discrete (versus global) disruptions caused by missense mutations has led us to question whether partial loss-of-function defects may account for some of the phenotypic heterogeneity characteristic of peripherin/rds-associated retinal diseases. To improve our understanding of protein domain structure and function, we have initiated an insertional mutagenesis approach similar to one taken previously for rhodopsin (28Borjigin J. Nathans J. J. Biol. Chem. 1994; 269: 14715-14722Abstract Full Text PDF PubMed Google Scholar). This report provides evidence in support of the hypothesis that the EC2 domain is particularly important for proper folding and subunit assembly of peripherin/rds. A series of 14 Phase I insertion mutants, each containing a unique Nar I endonuclease restriction site (5′-GGCGCC-3′), was constructed using subcloned regions of the WT peripherin/rds sequence as templates, synthetic oligonucleotide primers (TableI), and methods described previously (29Goldberg A.F. Molday R.S. Methods Enzymol. 2000; 316: 671-687Crossref PubMed Google Scholar). The presence of designed, and absence of spurious, mutations was confirmed by complete (single-stranded) DNA sequencing using a BigDye terminator cycle sequencing cycle kit (Applied Biosystems, Inc.). Mutagenized regions were returned to a WT peripherin/rds expression vector background by subcloning, and final constructs were confirmed by restriction mapping. Each Phase I mutation introduces a dipeptide (GA) into the WT protein sequence at the indicated position (see Fig.1).Table ISynthetic oligonuclueotides used for Phase I mutagenesisSynthetic oligonucleotide primers (shown) were employed to create 14 Phase I insertion mutants using a WT peripherin/rds template; each contains a unique Nar I endonuclease restriction site (underlined). Polymerase chain reaction methods and template subclones have been described previously (24Goldberg A.F. Loewen C.J. Molday R.S. Biochemistry. 1998; 37: 680-685Crossref PubMed Scopus (102) Google Scholar). Open table in a new tab Figure 1Sites of insertional mutation in peripherin/rds. The primary sequence (346 amino acids) and current folding topology model of bovine peripherin/rds are illustrated. Fourteen individual Phase I mutants (numbered) were constructed by the insertion of Nar I restriction sites at the breakpoints indicated; Phase I mutations add a Gly-Ala dipeptide to the WT sequence. Fourteen additional Phase II mutants were constructed by directional ligation of an epitope cassette coding for a nonapeptide FLAG epitope (DYKDDDDKA) into each Nar I site.View Large Image Figure ViewerDownload (PPT) Synthetic oligonucleotide primers (shown) were employed to create 14 Phase I insertion mutants using a WT peripherin/rds template; each contains a unique Nar I endonuclease restriction site (underlined). Polymerase chain reaction methods and template subclones have been described previously (24Goldberg A.F. Loewen C.J. Molday R.S. Biochemistry. 1998; 37: 680-685Crossref PubMed Scopus (102) Google Scholar). A series of 14 Phase II mutations was constructed by ligation of a FLAG epitope cassette (30Chubet R.G. Brizzard B.L. BioTechniques. 1996; 20: 136-141Crossref PubMed Scopus (79) Google Scholar) flanked by Nar I sticky ends (sense, 5′-CGACTACAAGGACGACGACGACAAGGC-3′; antisense, 5′-CGGCCTTGTCGTCGTCGTCCTTGTAGT-3′) into the Phase I mutants described. The presence, copy number, and orientation of the FLAG cassette were determined by DNA sequencing with a BigDye terminator cycle sequencing kit (Applied Biosystems, Inc.). Each of the Phase II mutants contains a nonapeptide insertion encoding a FLAG epitope (DYKDDDDKA) at the indicated position within the Phase I mutant sequences (see Fig.1). Anti-peripherin/rds monoclonal antibody C6 was a kind gift of Dr. John Saari and Dr. Krzysztof Palczewski of the Department of Ophthalmology at the University of Washington. Anti-rom-1 polyclonal antibody MUTT was generated by immunization of New Zealand White rabbits with an affinity-purified glutathioneS-transferase fusion protein. The coding sequence for the C-terminal hydrophilic domain of bovine rom-1 was amplified from a λGT-11 bovine retinal library and then cloned in-frame into the pGEX-2T expression vector. Bacterial fusion protein expression and affinity purification were performed essentially as described (31Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). Each antibody was characterized with respect to its specificity in Western blot analyses using authentic proteins from rod outer segment membranes, and recombinant proteins were expressed in COS-1 cells; no cross-reactivity was observed. The anti-peripherin/rds monoclonal antibody (MAb) C6 epitope was mapped using a series of synthetic peptides kindly provided by Dr. Kathleen Boesze-Battaglia, and it was found to reside within 15 amino acids of the full-length protein C terminus. It was not expected to be affected by any of the mutations examined in this report. Peptide competition was also used to confirm MAb C6 specificity for other immunochemical procedures, including immunofluorescence localization and immunoprecipitation studies not reported here. COS-1 cells (∼1 × 106/100-mm dish) were transfected with the FuGENE 6 reagent (Roche Diagnostics Co.) and 8 μg of the indicated expression plasmid essentially as suggested by the manufacturer. Detergent extracts were prepared using 1% Triton X-100 in phosphate-buffered saline at 48 h post-transfection essentially as described (30Chubet R.G. Brizzard B.L. BioTechniques. 1996; 20: 136-141Crossref PubMed Scopus (79) Google Scholar). Methods for Western blot analysis of recombinant peripherin/rds expression and disulfide bonding have been reported previously (23Goldberg A.F. Moritz O.L. Molday R.S. Biochemistry. 1995; 34: 14213-14219Crossref PubMed Scopus (97) Google Scholar). COS-1 cells (∼3 × 104/4-cm2 slide chamber) were transfected with FuGENE 6 (Roche Diagnostics Co.) and 1 μg of the indicated expression plasmid essentially as suggested by the manufacturer. Cells were fixed briefly with 4% paraformaldehyde, permeabilized with Triton X-100, and processed using MAb C6 and a secondary anti-mouse IgG covalently labeled with the Cy3 fluorophore (Amersham Pharmacia Biotech) essentially as described (23Goldberg A.F. Moritz O.L. Molday R.S. Biochemistry. 1995; 34: 14213-14219Crossref PubMed Scopus (97) Google Scholar). Images were collected with a Nikon Optiphot-2 microscope equipped with an epi-fluorescent illuminator and a SPOT RT digital imaging system (Diagnostic Instruments Inc., v3.0 software). S20, w estimates were made in a Beckman Optima TLX centrifuge using a TLA-55 rotor or in a Sorvall RC-M120EX centrifuge using an RP55-S rotor as described (29Goldberg A.F. Molday R.S. Methods Enzymol. 2000; 316: 671-687Crossref PubMed Google Scholar) but with the following modifications. Sucrose gradient fractions (2.1-ml) were collected by piercing tube bottoms in an offset fashion; particulate fractions were obtained by resealing punctured centrifuge tubes with laboratory film, adding 90 μl of 1× Laemmli sample buffer and vortexing vigorously. Digital analysis of chemiluminescent Western blots was performed using Scion Image software (Scion Corp.). Total peripherin/rds reactivity was calculated by pixel summation over soluble and particulate fractions. Regions of peripherin/rds consensus sequence predicted to form hydrophilic domains were evaluated for their tendency to form polypeptide secondary structure, and 14 sites within regions lacking strongly predicted structural motifs were identified (Fig.1). A series of 14 Phase I mutants, each containing a unique Nar I endonuclease restriction site, were constructed using synthetic oligonucleotides and the polymerase chain reaction. These mutations were designed to produce minimal perturbation to protein structure as they add only two relatively small, uncharged amino acids (GA) to the WT sequence. An additional series of 14 Phase II mutants, expected to be more highly disruptive, was constructed by ligation of a single copy FLAG epitope cassette (coding for a nonapeptide, DYKDDDDKA) into the Nar I site of each Phase I mutant. Each of the 28 mutant expression plasmids was transfected individually into COS-1 cells in culture. Protein expression was assessed by Western blot analysis using anti-peripherin/rds MAb C6. This reagent is useful as a measure of full-length protein expression because it recognizes a 15-amino acid epitope at the C terminus of peripherin/rds. Although none of the insertional mutations interrupt or lie immediately adjacent to the MAb C6 epitope, it was not known a priori whether the antibody would react with all mutants. In fact, expression of each of the 14 Phase I mutants is detected by MAb C6 in COS-1 cells. Similar to WT peripherin/rds, the mutants typically migrate as closely spaced doublets under denaturing and reducing conditions (Fig. 2 A). Moreover, peripherin/rds reactivity is also observed in extracts from COS-1 cells transfected with each of the 14 Phase II mutants (Fig.2 B). The Phase II mutants migrate somewhat more slowly than the WT protein and show a greater variation in electrophoretic mobility. The added mass and charge of the FLAG epitope insertion, heterogeneous post-translational modification, and/or residual secondary structure may be responsible for the observed differences. Identical results were obtained when Phase II mutant expression was assayed by probing Western blots with the anti-FLAG MAb M2 (not shown). Although we expected that the relatively large insertions introduced into the Phase II mutants might cause degradation and absent or reduced protein expression, reproducible differences in expression levels between the Phase I and Phase II mutants were not observed. These results demonstrate that neither minor (uncharged dipeptide) nor more major (charged hendecapeptide) insertions at any of 14 sites distributed throughout the primary sequence prevent peripherin/rds protein expression in COS-1 cells. WT peripherin/rds is extracted from vertebrate retina and COS-1 cells both as monomeric and disulfide-bonded dimeric forms, as assayed by nonreducing Western blot analysis. Abnormal disulfide bonding has been associated with several instances of human retinal disease. We therefore examined the mobility of each insertional mutant by Western analysis in the absence of added reducing agent. Eleven of the 14 Phase I mutants appear roughly similar to WT; they migrate primarily as both monomeric and dimeric forms (Fig.3 A). In contrast, three Phase I mutants (IM5, IM7, and IM11) show a banding pattern dissimilar from WT (Fig. 3 A, lanes 5, 7, and11). These mutants exhibit a strong tendency to form large aggregates that remain trapped in the stacking gel. Essentially identical results were obtained for the Phase II series of mutations (Fig. 3 B). The combined data indicate that neither Phase I nor Phase II mutations at any of 11 insertion sites prevents normal disulfide bonding but that a mutation at any one of three sites (IM5, IM7, or IM11) disrupts normal disulfide bonding and destabilizes protein structure. WT peripherin/rds is assembled as a tetrameric protein in both retinal photoreceptors and transfected COS-1 cells. Tetramerization appears to be essential for function as impaired subunit assembly has been linked to dysmorphic OSs in mice and several forms of retinal degeneration in humans (24Goldberg A.F. Loewen C.J. Molday R.S. Biochemistry. 1998; 37: 680-685Crossref PubMed Scopus (102) Google Scholar, 27Goldberg A.F. Molday R.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13726-13730Crossref PubMed Scopus (105) Google Scholar). We have previously developed a reliable method for the assay of tetramer formation in COS-1 by characterizing protein sedimentation velocity (29Goldberg A.F. Molday R.S. Methods Enzymol. 2000; 316: 671-687Crossref PubMed Google Scholar) and apply it here to determine whether any of the insertional mutations affect this process. Phase I and Phase II mutants were centrifuged through sucrose density gradients, and fractionated gradients were analyzed by Western blotting using MAb C6. This approach generated sedimentation profiles that fall into four main classes; raw data for particular Phase II mutants typical of each class are presented (Fig.4, B–E). A representative sedimentation profile for WT peripherin/rds is given for comparison (Fig. 4 A). Sedimentation coefficients (S20, w values) calculated for each hydrodynamically distinct species observed are presented in TableII.Table IISedimentation coefficients (S 20, w) of peripherin/rds mutantsPhase IPhase IIWT5.4 ± 0.1 (3)2-aDetermined in a previous study (24).2-bMean ± S.D. from nsamples (number in parentheses).5.4 ± 0.1 (3)2-aDetermined in a previous study (24).IM15.5 (2)5.7 (2)IM25.5 (2)5.5 ± 0.3 (4)IM35.5 (2)5.8 (2)IM45.5 (2)5.6 (2)IM5P (2)P (3)IM65.4 (2)5.6 (2)IM7P (2)P (2)IM84.5/5.32-cIn half the experiments, reactivity sedimented with a mean of 4.5, in others it sedimented with a mean of 5.3. (4)3.9 ± 0.2 (3)IM95.3 ± 0.3 (3)5.7 (2)IM104.2 ± 0.3 (3)3.8 ± 0.2 (3)IM115.6 (2)/P2-dIn each experiment, approximately half the reactivity was found in the pellet.Variable (6)/P2-eThe proportion of reactivity found in the pellet varied (10–90%) between experiments.IM125.6 (1)5.5 (2)IM135.4 (2)5.7 (2)IM145.6 (2)5.6 (2)Sedimentation coefficients (S20, w) for peripherin/rds variants expressed in COS-1 cells were determined as described under “Experimental Procedures.” The number of independent transfection/sedimentation trials performed to obtain each value is given by n; standard deviations are given where appropriate. Mutants that were recovered from the particulate fraction are indicated (P).2-a Determined in a previous study (24Goldberg A.F. Loewen C.J. Molday R.S. Biochemistry. 1998; 37: 680-685Crossref PubMed Scopus (102) Google Scholar).2-b Mean ± S.D. from nsamples (number in parentheses).2-c In half the experiments, reactivity sedimented with a mean of 4.5, in others it sedimented with a mean of 5.3.2-d In each experiment, approximately half the reactivity was found in the pellet.2-e The proportion of reactivity found in the pellet varied (10–90%) between experiments. Open table in a new tab Sedimentation coefficients (S20, w) for peripherin/rds variants expressed in COS-1 cells were determined as described under “Experimental Procedures.” The number of independent transfection/sedimentation trials performed to obtain each value is given by n; standard deviations are given where appropriate. Mutants that were recovered from the particulate fraction are indicated (P). Neither Phase I nor Phase II insertional mutations affected velocity sedimentation profiles at most (9 of 14) sites, including IM1, IM2, IM3, IM4, IM6, IM9, IM12, IM13, and IM14. All were essentially indistinguishable from WT (Fig. 4, A and B and Table II). These sites are distributed throughout the polypeptide primary sequence (Fig. 1), and their lack of effect demonstrates that subregions within every predicted hydrophilic domain can tolerate both small (uncharged) and large (highly charged) insertional mutations without global disruption of WT structure. In contrast, either large or small insertions into any one of five sites, all contained within the EC2 domain, resulted in largely non-WT sedimentation behavior. Insertions into two sites, IM5 and IM7, caused increased sedimentation velocities and recovery of the aggregated mutants from the particulate fraction (Fig. 4 B and TableII). These effects illustrate sites at which insertional mutations sufficiently reduce protein structural stability to generate global misfolding. More interestingly, mutants at IM8 and IM10 sediment with a velocity substantially less than that of WT (Fig. 4 Dand Table II). Mutations at these sites result in sedimentation coefficients that are more characteristic of peripherin/rds dimers than tetramers, and they appear to disrupt normal subunit assembly without impairing protein folding. Finally, IM11 mutants revealed more complex and variable behavior than insertions in other sites (Fig.4 E). Phase I (IM11) insertions generated both grossly misfolded and WT species, in an approximately equal ratio, whereas Phase II (IM11) insertions produced variable amounts of aggregated protein and hydrodynamic species of variable (non-WT) mobility (Fig.4 E and Table II). In sum, these results demonstrate that insertional mutations at five of seven sites within EC2 cause measurable changes in protein structure; these insertions produced either global structural disruption (IM5 and IM7), altered subunit assembly (IM8 and IM10), or both (IM11). WT peripherin/rds expressed in COS-1 cells is retained largely within intracellular membranes; a previous study (23Goldberg A.F. Moritz O.L. Molday R.S. Biochemistry. 1995; 34: 14213-14219Crossref PubMed Scopus (97) Google Scholar) reported a distinctly perinuclear subcellular distribution and suggested that the protein was localized within the Golgi apparatus. Our more recent co-localization studies utilize a Golgi-specific marker and support this interpretation. 2N. Khattree and A. Goldberg, unpublished observations. Because quality control mechanisms are known to prevent misfolded membrane proteins from exiting the endoplasmic reticulum and entering the Golgi apparatus (32Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1066) Google Scholar), we speculated that mutations that alter peripherin/rds folding and subunit assembly might also affect its subcellular localization. We used immunofluorescence microscopy to examine whether insertions in EC2 that were disruptive for protein folding or assembly also affected subcellular localization. It should be noted that flu" @default.
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• W2000429226 date "2001-11-01" @default.
• W2000429226 modified "2023-09-28" @default.
• W2000429226 title "Folding and Subunit Assembly of Photoreceptor Peripherin/rds Is Mediated by Determinants within the Extracellular/Intradiskal EC2 Domain" @default.
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• W2000429226 doi "https://doi.org/10.1074/jbc.m107511200" @default.
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