Figure 7.

Extracellular PHB levels of cells expressing the 5S-G and Y826G mutants are significantly reduced.

Panel A: PHB signals on the cell surface obtained by immunocytochemical analyses of the polymer with anti-PHB-IgG on non-permeabilized cells detected with confocal microscopy (for details see supplementary methods section). The images were obtained with a Zeiss (Oberkochen, Germany) LSM-510 confocal microscope (60X objective). HEK-293 cells were transiently transfected with the wild type TRPM8 (1 μg), the 5S-G (1 μg), and the Y826G (1 μg) mutants along with GFP (0.2 μg) for detection purposes.

Panel B: Summary of intensities of the PHB signals on PM measured for the wild type TRPM8 and the mutants 5S-G and Y826G. Panel C: The surface expression of the protein is not altered. The wild type TRPM8 and the mutants (5S-G, Y826G) were biotinylated and captured with streptavidin beads as described under the supplementary methods section. Panel D: Arithmetic means of the relative density of the proteins in the membrane fraction of wtTRPM8 and the 5S-G and Y826G mutants, n = 3. Panel E: Cartoon of a model for the PHB-TRPM8 complex with PHB attachment to the extracellular peptide LHSSNHSSLYSGR (817–829) and supported by a covalent bond to S827 and by hydrophobic interactions with Y826 and other hydrophobic residues in the region; red spheres show putative intracellular PHBylation sites of TRPM8.