Figure 1
From: Cargo binding promotes KDEL receptor clustering at the mammalian cell surface
H/KDEL-cargo binding to the mammalian cell surface induces receptor cluster formation.
(A) (top) Schematic outline of the fluorescent model cargo eGFP-RTAHDEL consisting of the cytotoxic A-subunit of ricin (RTA), mammalian enhanced GFP (eGFP) and a C-terminal (His)6-Tag for purification. (bottom) eGFP-RTA lacking a KDELR binding motif served as negative control. (B) Cell surface biotinylation of mammalian KDELR1. HeLa cells were transiently transfected with KDELR1 (Erd2.1-V5 (+)) or an empty vector (−) and cultivated for 48 h. Cell surface proteins were biotinylated by treatment with (+) or without (−) Sulfo-NHS-SS-Biotin and purified with streptavidin beads. Whole cell lysates (input) served as control to determine the total amount of Erd2.1-V5 (detected with anti-V5), while β-actin and Na+/K+ ATPase served as cytosolic and plasma membrane marker proteins, respectively. Membrane fraction (surface) illustrates the total fraction of proteins at the cell surface. (C) (bottom) Schematic outline of α-bungarotoxin (Btx) cell surface binding. HeLa cells expressing a KDELR variant in which a Btx binding site (BBS) was inserted between positions T114 and P115 of c-myc-tagged KDELR1 (Erd2.1) were treated with Alexa488-labeled α-Btx. As Btx is incapable to cross the mammalian PM, any physical interaction between Btx and BBS can only occur if KDELR1 is present in the PM. (top) Confocal laser scanning microscopy of HeLa cells transfected with pERD2.1-BBS-cmyc or an empty vector control and treated with 10 μg/ml Alexa488-labeled α-Btx. (D) In vivo toxicity of eGFP-RTAH/KDEL against HeLa cells. Cell viability (N = 3, n = 5 replicates) was determined after 48 h in the presence or absence of 160 μg/ml of the indicated RTA variant (Mock, PBS buffer). Mean values and standard deviations are displayed (***P < 0.001, t test). (E) Fluorescence microscopy of cargo binding at the cell surface. HeLa cells were treated with 160 μg/ml eGFP-RTAH/KDEL or eGFP-RTA for 5 min and cargo binding was analyzed after 10 washing steps. (F) Live cell imaging (45 frames/h) of HeLa cells treated with 160 μg/ml eGFP-RTAHDEL. Three representative time points (0, 30, 60 min) are shown. (G) Temporal evolution of the density of cargo signals at the surface of HeLa cells. The accumulation of fluorescent signals is shown after treatment with 160 μg/ml eGFP-RTAHDEL or eGFP-RTA. The symbols represent the optimal signal-to-noise ratio in image analysis. The error bars reflect the variation range of signal intensity for different threshold values of image analysis parameters. The functional form only weakly depends on the choice of the threshold values.
