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JC Krouse, FP Stefano, K Boesze-Battaglia; Mechanism Of Enhanced Fusion By A P296T Human Peripherin/rds Mutant . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1396.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The C-terminus of peripherin/rds promotes membrane fusion through a fusion peptide domain, residues 311-325. A proline to threonine mutation upstream of the fusion peptide domain at position 296 results in enhanced fusion. The work presented herein address the mechanism by which this fusion enhancement occurs. Methods: A series of mutations of the proline at residue 296 were generated in the pGemex-2 vector containing a Not 1 cloned 1.1Kb fragment of human peripherin/rds cDNA. WT and mutant proteins were N-terminal tagged using a pcDNA 3.1 His B vector, allowing for antibody detection (Xpress epitope tag) and purification on a Ni+2 column (polyhistidine tag) and transiently transfected into MDCK and COS-1 cells. Peripherin/rds-lipid recombinants were prepared using detergent dialysis with vesicles composed of PC:PS:cholesterol (4:4:1 mole ratio). Fusion was measured using resonance energy transfer techniques. Peripherin/rds localization was determined with specific marker proteins using immunofluorescence microscopy. Results: The WT and P296A, P296L, P296E mutant peripherin/rds were expressed as 42 kDa monomers detected using western blot analysis with anti-Xpress antibody under denaturing conditions. The purified proteins were recombined into liposomes that fused with R18 ROS PM, confirming a viable functional assay for the proteins. WT-Xpress peripherin/rds showed cellular localization to both intracellular and plasma membranes. The P296T mutant initially localized to intracellular membranes, but upon confluence appeared mostly in the plasma membrane. Conclusions: These results suggest that an area upstream of the fusion peptide domain, analogous to SNARE-pin like regions in other fusion protein may aid in promoting the fusion competency of peripherin/rds. The C-terminus of peripherin/rds may provide the localization signal for incorporation of peripherin/rds into the plasma membrane of MDCK cells.
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