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AF X Goldberg, LM Ritter, N Khattree, K Boesze-Battaglia; Importance Of C-terminal Charged Residues For Subunit Assembly And Fusogenicity Of Peripherin/rds . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1397.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the importance of charged residues within a C-terminal region of peripherin/rds for: protein folding, subunit assembly, subcellular localization, and fusogenicity. Method: Site-directed mutagenesis was utilized to neutralize three charged residues residing within a region of peripherin/rds proposed to form an amphipathic helix. Each residue was altered individually, and in combination, to generate a series with decreasing amphipathic character. Polypeptides were expressed in two contexts: 1) as full-length polytopic proteins (in COS-1 cells), and 2) as GST fusion proteins containing only hydrophilic C-terminal domains (in E. coli). Western blotting, immunocytochemistry, sedimentation velocity analysis, size exclusion chromatography, membrane depolarization and fluorescent lipid quenching methods were employed to assay mutants for expression, subcellular localization, subunit assembly, stoichiometry, membrane interaction and fusogenicity. Results: A set of seven C-terminal mutants were expressed as full-length and fusion proteins in COS-1 cells and E.coli respectively. Although subunit assembly and subcellular localization appear similar to wild-type for all mutants examined thus far, three of seven mutants do not appear to destabilize membranes, and none appear able to catalyze membrane fusion in vitro. Conclusion: Mutations that replace charged residues with neutral amino acids within a predicted amphipathic helix in the peripherin/rds C-terminus disrupt fusogenicity, but not biosynthesis, subunit assembly, or subcellular localization. These results demonstrate that particular aspects of peripherin/rds structure and function are segregated within discrete protein domains.
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