Heterologous Expression of WT and Mutant Photoreceptor Peripherin/rds in Madin Darby Canine Kidney Cells: an Assessment of Fusogenic Function
Rod Outer Segment
Peripherin/rds is proposed to function as a fusion protein within the rod outer segment and a fusion domain has been mapped to amino acids 311–325 within the C-terminus. To map regions within peripherin/rds required for membrane fusion a series of C-terminal mutants was analyzed. Madin Darby canine kidney cells were transiently transfected with an Xpress or FLAG epitope tagged peripherin/rds (wt) and three mutants of peripherin/rds. The mutants selected were a P296T mutant (replacement of the proline at position 296 with a threonine) and two C-terminal deletion mutants (one lacking the terminal 10 amino acids, Δ10 and one lacking the terminal 50 amino acids, Δ50). The wt protein, the P296T and Δ10 mutants were detected on SDS–PAGE as 84 kDa dimers, that resolved into 38–42 kDa monomers under reducing conditions. The Δ50 mutant showed a slightly increased mobility. The cellular localization of mutants differed from that of wt peripherin/rds. The wt Xpress-human and wt FLAG-bovine peripherin/rds were localized to both intracellular and plasma membranes. In contrast, the C-terminal deletion mutants were localized only to the intracellular membrane. The P296T mutant presented a still different pattern: initially the protein localized to intracellular membranes. Upon confluence, however, the localization appeared to become predominantly plasma membrane. To assess the fusion activity of the proteins, the cell membranes were fractionated using sucrose density gradient centrifugation and the various fractions identified based on immunoreactivity in Western blot analysis with Golgi (anti-rab 6) or plasma membrane (anti-ZO-3) specific marker proteins. All membrane fractions were assayed for fusion with ROS plasma membrane vesicles. The plasma membrane enriched fractions (isolated at densities of 1·08 and 1·125 g ml−1) containing tagged peripherin/rds and the Δ10 mutant promoted membrane fusion with ROS plasma membrane vesicles. In contrast, fusion was not detected with plasma membrane vesicles from mock-transfected cells or the Δ50 peripherin/rds deletion mutant. Fusion was enhanced in a less dense fraction enriched in the P296T mutant (isolated from the 1·04/1·02 interface) relative to wt. Fusion was dependent on the presence of peripherin/rds in the membranes and could be inhibited with trypsinolysis and competition studies with the bovine fusion peptide, PP-5. Peptide competition suggests that the fusion domain of human peripherin/rds is most likely identical to that characterized in bovine and corresponds to amino acid residues 312–326. The C-terminal deletion mutants have allowed us to predict the minimal region of the C-terminus necessary for fusion to include residues starting at number 335. In addition a second region important in the formation of a fusion competent peripherin/rds has been mapped to a region upstream of the fusion peptide domain.