An amphipathic helix facilitates the membrane binding properties of BFSP1 and its caspase-generated C-terminal domain

Abstract

Background: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aqua- 21 porin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is 22 myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic 23 analyses suggested that the sequences 434-452 were a-helical and amphipathic. Methods and Re- 24 sults: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an 25 intrinsically disordered to a more a-helical conformation for the residues 434-467. Recombinantly 26 produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as deter- 27 mined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non- 28 lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to 29 cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compart- 30 ments such as the nuclear and mitochondrial membranes were negative. The N-terminal myristoy- 31 lation of the amphipathic helix appeared not to change either the lipid affinity or membrane locali- 32 sation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it 33 did appear to enhance its helical content. Conclusions: These data support the conclusion that C- 34 terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane 35 binding properties via an adjacent amphipathic helix