An inducible amphipathic helix within the intrinsically disordered C terminus can participate in membrane curvature generation by peripherin-2/rds.
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 andXenopus laevisphotoreceptors. We also studied a soluble form of the full-length ∼7-kDa cytoplasmic C terminus in cultured cells and purified fromEscherichia coliThe α-helical motif was not required for protein biosynthesis, tetrameric subunit assembly, tetramer polymerization, localization at disk rims, interaction with GARP2, or the generation of membrane curvature. Interestingly, however, loss of the helical motif up-regulated membrane curvature generationin 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 the outer segment disk and suggests that the protein's C terminus may modulate membrane curvature-generating activity present in other protein domains.
From the Eye Research Institute, Oakland University, Rochester, Michigan 48309 and.
Michelle L
ML
Milstein
From the Eye Research Institute, Oakland University, Rochester, Michigan 48309 and.
Victoria A
VA
Kimler
the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421.
Chiranjib
C
Ghatak
the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421.
Alexey S
AS
Ladokhin
From the Eye Research Institute, Oakland University, Rochester, Michigan 48309 and goldberg@oakland.edu.
Andrew F X
AFX
Goldberg
D000818:Animals; D030801:Animals, Genetically Modified; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry*; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry*; D009154:Mutation; D064531:Peripherins / Q000737:chemistry* / Q000502:physiology; D010743:Phospholipids / Q000737:chemistry; D000072417:Protein Domains; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D014982:Xenopus laevis
D054815:Cyclic Nucleotide-Gated Cation Channels; D064267:Intrinsically Disordered Proteins; D064531:Peripherins; D010743:Phospholipids; C578919:glutamic acid-rich protein-2, human
amphipathic helix; intrinsically disordered; membrane curvature; membrane protein; outer segment; photoreceptor; retina; retinal degeneration; tetraspanin