Title
An Inducible Amphipathic Helix Within the Intrinsically Disordered C-terminus is not Required for Protein Biosynthesis, Trafficking, or GARP2 Interaction, but can Participate in Membrane Curvature Generation by Peripherin-2/rds.
Author
Michelle L. Milstein, Victoria A. Kimler, Chiranjib Ghatak, Alexey S. Ladokhin, Andrew F.X. Goldberg
Year
2017
Journal
The Journal of Biological Chemistry
Abstract
Peripherin-2/rds is required for biogenesis of vertebrate photoreceptor outer segment organelles. Its localization at the high curvature rim domains of outer segment disk membranes suggests that it may act to shape these structures; however, the molecular function of this protein is not yet resolved. Here, we apply biochemical, biophysical, and imaging techniques, to elucidate the role(s) played by the protein's intrinsically disordered C-terminal domain, and an incipient amphipathic α-helix contained within it. We investigated a deletion mutant lacking only this α-helix, in stable cell lines and X. laevis photoreceptors. We also studied a soluble form of the full-length ~7 kDa cytoplasmic C-terminus, in cultured cells and purified from E. coli. The α-helical motif was not required for protein biosynthesis, tetrameric subunit assembly, tetramer polymerization, localization at disk rims, interaction with GARP-2, or the generation of membrane curvature. Interestingly however, loss of the helical motif upregulated membrane curvature generation in cellulo, introducing the possibility that it may regulate this activity in photoreceptors. Furthermore, the incipient α-helix (within the purified soluble C-terminus) partitioned into membranes only when its acidic residues were neutralized by protonation. This suggests that within the context of full-length peripherin-2/rds, partitioning would most likely occur at a bilayer interfacial region, potentially adjacent to the protein's transmembrane domains. In sum, this study significantly strengthens the evidence that peripherin-2/rds functions directly to shape the high curvature rim domains of outer segment disks, and suggests that the protein's C-terminus may modulate membrane curvature generating activity present in other protein domains.
Instrument
J-720
Keywords
Circular dichroism, Secondary structure, Chemical stability, Vesicle interactions, Biochemistry