Folding and subunit assembly of photoreceptor peripherin/rds is mediated by determinants within the extracellular/intradiskal EC2 domain: implications for heterogeneous molecular pathologies

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.

Fig.1.

Sites 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.

Fig.2.

Assay of insertion mutant protein expression by Western blot analysis. Detergent extracts of transfected COS-1 cells were reduced with β-mercaptoethanol, run on a 10% SDS polyacrylamide gel and then electroblotted onto Immobilon-P membrane and probed with anti-peripherin/rds MAb C6. All 14 Phase I (A) and 14 Phase II (B) insertion mutants express peripherin/rds. Phase II mutants display reduced mobility relative to WT due to the addition of a FLAG epitope; slight variations in apparent molecular mass (MW) may be a function of heterogeneous post-translational modification and/or residual secondary structure.

Fig.3.

Assay of insertion mutant disulfide bonding by nonreducing Western blot analysis. Detergent extracts of transfected COS-1 cells were treated with N-ethylmaleimide to block free thiols, subjected to nonreducing 10% SDS-PAGE, and then electroblotted onto Immobilon-P membrane and probed with anti-peripherin/rds MAb C6. All 14 Phase I (A) and 14 Phase II (B) insertion mutants are observed as several species. Three mutants show a pronounced tendency relative to WT to form higher order aggregates: IM5, IM7, and IM11. Similar results are observed for the Phase I and Phase II mutations.

Fig.4.

Assay of Phase II mutant subunit assembly by velocity sedimentation. Detergent extracts from transiently transfected COS-1 cell cultures were sedimented under reducing conditions in 5-20% (w/w) sucrose gradients. Fractionated gradients and particulate fractions (P) were assayed for peripherin/rds by Western blot analysis with MAb C6. Chemiluminescent blots and corresponding plots generated by image analysis are shown (A-E); four classes of sedimentation profile are evident. WT peripherin/rds (A) sediments as a single major peak characteristic of a tetrameric stoichiometry (24). Most insertional mutants, including IM9 (C), gave similar results, indicating that their subunit assembly is normal. Two mutants, including IM10 (D), displayed sedimentation profiles more characteristic of dimeric peripherin/rds (37). Insertions at two sites, including IM7 (B), caused gross aggregation as protein was only recovered from the particulate fraction (P). Insertion at one site, IM11 (E), was characterized by variable behavior. Both dimeric and aggregated species are evident; this site appears to destabilize protein structure in a significant yet incomplete fashion. Sedimentation coefficient estimates for all 28 Phase I and Phase II mutants were calculated as described previously (24) and are reported in Table II.

Fig.5.

Subcellular localization of Phase II insertion mutants by indirect immunofluorescence microscopy. COS-1 cells transiently transfected with the indicated mutant expression vectors were fixed with paraformaldehyde, permeabilized with Triton X-100, and labeled with anti-peripherin/rds MAb C6. WT peripherin/rds (A) has previously been localized to internal membranes, a perinuclear localization pattern typical of distribution within Golgi membranes is apparent. Insertional mutations that do not result in grossly misfolded protein, including IM2 (B), IM8 (E), IM9 (F), and IM10 (G), display similar perinuclear distributions. Insertions that do result in grossly misfolded protein, including IM5 (C), IM7 (D), and IM11 (H), produce larger and more numerous fluorescent blobs that are distributed less focally and are characteristic of localization within the endoplasmic reticulum.

Fig.6.

Molecular consequences of insertional mutations on peripherin/rds structure. Combined findings from investigations of disulfide bonding, subunit assembly, and subcellular localization are integrated to summarize current conclusions. The majority of peripherin/rds polypeptide sequence can tolerate either small uncharged (dipeptide) or larger, negatively charged (hendecapeptide) insertions without measurable effects on protein folding or tetrameric subunit assembly (white ovals). Mutations that do affect protein structure all reside within EC2, the second extracellular/intradiskal domain. Insertions caused either grossly misfolded protein (black ovals) or dimeric (versus tetrameric) subunit assembly (gray ovals). One site, IM11, produced each effect at variable ratios.