Fig. 4. Rop1 and its human homolog REEP1 generate high membrane curvature.
a SBP-tagged sjRop1 and sjYop1 from S. japonicus were expressed in E. coli. Cell lysates were subjected to ultracentrifugation and the membrane (M) and non-sedimentable (NS) fractions analyzed by SDS-PAGE, followed by blotting with fluorescently labeled streptavidin. b sjRop1 of the NS fraction was bound to streptavidin beads, the tag was removed with TEV protease, and the eluted material subjected to gel filtration. The peak fraction was analyzed by Coomassie-blue staining (left inset) and negative-stain EM (right inset). c Diameters of Rop1 particles compared to those of lipoprotein particles (LPP) generated in an analogous manner by Xenopus laevis REEP5 (XeREEP5)5. *** indicates a significant difference with p-value 2.1 × 10−32, calculated from a two-sample t test with unequal variance. d sjYop1, sjRop1, human REEP1 (hsREEP1) or the indicated mutants were purified from the membrane fraction and mixed with liposomes. The detergent was removed, and the reconstituted material was analyzed by negative-stain EM. Scale bar, 100 nm. The panels on the right show the domains of sjRop1. The APH is shown in gray with deleted regions indicated. Predicted breaks in the APH are indicated by vertical lines. e Diameters of structures seen in (d). For tubules, the largest width is given and for irregular structures, the largest dimension. *** indicates significant differences with p-values < 0.001, calculated from a two-sample t test with unequal variance. The exact p-values are listed in the Source Data file. f Reconstituted material as in (d) was subjected to flotation in a Nycodenz gradient. Fractions were analyzed by Coomassie-blue staining and protein abundance in individual fractions is expressed as percentage of total protein.