Fig. 2: Vesicle localization of REEP1-4 proteins is dependent on their APH-C.
From: The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment
a U2OS cells stably expressing the ER marker Sec61β fused to red fluorescent protein (RFP-Sec61β) were transfected with hemagglutinin (HA)-tagged REEP1 carrying the disease mutation Δ113-201, immunostained with anti-HA (αHA) antibodies, and imaged by confocal fluorescence microscopy. Right panel shows an overlay between αΗΑ (green) and RFP-Sec61β (magenta). Scale bar, 2 µm. b As in (a), but with cells transfected with the REEP1 disease mutant Δ102-139. c As in (a), but with cells transfected with the REEP2 disease-linked mutant Δ111-252. d Pearson’s correlation coefficients (r) measuring colocalization of RFP-Sec61β with wild-type or APH-C disease mutants of HA-tagged REEP1 or REEP2. Shown are means and standard deviations. n, REEP1wt, 153; REEP1Δ102-139, 164; REEP1Δ113-201, 166; REEP1Δ150-201,166; REEP1L107P, 168; REEP1L118R, 171; REEP1N127D, 163; REEP2WT, 106; REEP2 Δ111-252, 102 cells. P-values for REEP1 were calculated using one-way ANOVA analysis, multiple comparisons (Dunnett’s method). REEP2 p-values were calculated using an unpaired two-tailed t test. ****p < 0.0001; ns, not significant. Exact p-values are listed in the Source Data file. e As in (a), but in cells transfected with the REEP1-HA constructs carrying the L107P disease mutation, or putative disease mutations L118R or N127D. f The stabilities of C-terminal REEP1-HA mutants were analyzed by cycloheximide (CHX) chase. Cells transfected with wild-type or mutant HA-tagged REEP1 were collected at timepoint 0 or after CHX treatment for 6 h, and lysates were analyzed by immunoblotting with αHA and anti-tubulin (αtub) antibodies. g Schematic of REEP1 domain organization and APH-C prediction. C-terminal REEP1 point mutations that cause relocalization to the bulk ER are circled in red. Amphipathic helices were predicted using heliquest (https://heliquest.ipmc.cnrs.fr/)70. The arrows indicate the hydrophobic moments. Note that all mutations are either helix breakers or reside along the APH-C’s hydrophobic face. h REEP4 knockout U2OS cells stably expressing mEm-fused REEP4 (I107P) (top panels) or wild-type REEP4 (bottom panels) and transfected with RFP-Sec61β were imaged by confocal microscopy. Right panels show overlays between mEm (green) and RFP-Sec61β (magenta). Scale bars, 2 µm.
