Fig. 1: REEP1 and REEP4 localize to vesicles in mammalian cells.
From: The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment
a Alphafold-predicted models of the monomer and homodimer of human REEP1 and REEP5. Transmembrane (TM) segments are numbered and colored in blue. Amphipathic helices at the N- or C-termini (APH-N or -C, respectively) are in sienna. The second monomer in the dimer is shown in gray. b Endogenous REEP1 localization in SaOS2 cells stably expressing the ER marker Sec61β fused to near-infrared fluorescent protein (iRFP-Sec61β). Cells were analyzed by indirect immunofluorescence using anti-REEP1 (αREEP1) and anti-alpha tubulin (αtubulin) antibodies, and imaged using confocal fluorescence microscopy. Shown are whole cells and enlargements of the boxed region. Right column shows overlays of αREEP1 (green) and iRFP-Sec61β (magenta). Bottom two rows show cells after 50 μM nocodazole treatment. c Endogenous REEP4 localization was determined in U2OS cells stably expressing mScarlet-Sec61β (magenta) by immunostaining with anti-REEP4 antibodies (αREEP4, green). Right panels show overlays, and bottom two rows show cells after nocodazole treatment. d U2OS cells stably co-expressing REEP1-mEmerald (REEP1-mEm, green) and the ER marker mScarlet-Sec61β (magenta) were imaged using confocal fluorescence microscopy. Right panels show overlays. Bottom panels show magnifications of the boxed region. e As in (d), but with cells stably expressing REEP5-mEmerald (REEP5-mEm) and transiently transfected with mScarlet-Sec61β. f Membrane fractionation of U2OS cells stably expressing REEP1-mEm. Cell lysates were fractionated into supernatant (S/N) and membrane pellet by ultracentrifugation, and the pellet was solubilized in 1% dodecylmaltoside (DDM) detergent. Samples were analyzed by SDS-PAGE and immunoblotting with antibodies against mEmerald (αGFP), REEP5 (αREEP5), the membrane protein calnexin (αCNX), and cytosolic alpha tubulin (αtub). Arrowhead denotes full-length REEP1-mEm; asterisk, mEm fragment. g U2OS cells stably expressing REEP1 fused to ascorbate peroxidase (REEP1-APEX) were fixed, treated with diaminobenzidine and hydrogen peroxide (+DAB/H2O2), and analyzed by thin section electron microscopy. Arrowheads point to electron-dense, circular structures in +DAB/H2O2 samples. er, endoplasmic reticulum. Scale bars, 50 nm. h As in (g), but without DAB/H2O2 treatment (-DAB/H2O2). i, Quantification of the diameters of REEP1-APEX positive structures observed in (g). Shown are the mean and standard deviation. n, 186. For all scale bars (b–e), whole cell, 10 µm; magnification, 2 µm.
