Self-association of MBP molecules via hydrophobic interactions is required for its function.

(A) Quantification of FRET efficiency in PtK2 cells expressing GFP-Tm10 (Donor) and mCherry-GyPTM (Acceptor) both harboring at the C-terminal end either wild-type MBP or MBP F→S. While Tm10 represents the transmembrane domain of Tmem10, GyPTM represents the mutated monomeric transmembrane domain of the glycophorin protein. Bars indicate mean ± SD (n = 20 cells, *p<0.05, ANOVA). (B) Comparison of interaction forces between wild-type MBP or F→S mutant molecules pre-adsorbed, both to the mica surface and AFM tip. Inset shows the schematic depiction of shape of the curve as cantilever tip approaches the sample surface (1), as tip touches the surface (2), and as tip is retracted from the sample surface (3). Histogram of peak force measured for MBP (black), MBP F→S (red), and buffer (green). (C) Representative images of a biomimetic assay in which MBP or MBP F→S is sandwiched between SLBs (inner myelin leaflet lipid composition) and GUVs (PC∶PS in 3∶1 mole%). Scale bar, 10 µm. (D) Quantification of percentage GUV bursting. Bars show mean ± SEM (n = 3 experiments, ***p<0.001, t test). (E) Shiverer cells at 0 DIV were infected with AAV2 viral particles expressing either wild-type MBP (MBP-HA) or F→S mutant (MBP F→S-HA) containing a C-terminal HA-tag. At 6 DIV, cells were immunostained for CNPase and the HA tags. Expression of MBP-HA induces the depletion of CNPase from regions within the sheets, whereas the F→S mutant fails in extruding CNPase despite entering the sheets of shiverer cells. Enlarged view of the selected regions in merged images is shown on the right side. Scale bar, 10 µm.