Figure 2. VMP1 promotes Ca 2 transport... | Rockefeller University Press

Figure 2.

VMP1 promotes Ca2+transport across membranes. (A) C-terminal amino acid sequence of VMP1. Lysine residues in the Golgi-to-ER retrieve motif are labeled in blue and the mutated alanine was labeled in red. (B) The interactions between RFP, VMP1, or VMP1-K404/406A with COPI were examined by coimmunoprecipitation. Irrelevant lanes were removed and a dash line is shown. (C) Subcellular localization of FLAG-tagged (N-terminal) VMP1 and VMP1-K404/406A was examined by immunofluorescence. Plasma membrane was labeled by WGA. (D) Putative topology of VMP1 on ER. (E) Putative topology of VMP1-K404/406A on plasma membrane. (F) An illustration of Ca2+ transport mediated by VMP1 on ER and VMP1-K404/406A on plasma membrane. (G and H) Ca2+ influx in HEK293T cells transfected with empty vector, VMP1, or VMP1-K404/406A was measured by flow cytometry with 8 mM CaCl2. (I) Ca2+ influx in HEK293T or STIM1-KO HEK293T cells transfected with empty vector, VMP1, or VMP1-K404/406A was measured by flow cytometry with 8 mM CaCl2. (J and K) Whole-cell patch-clamp traces of STIM1-KO HEK293T cells transfected with pCMV-GFP or pCMV-VMP1-K404/406A. Final concentration of 20 mM CaCl2 was added to bath solution. The holding potential was −100 mV. (B and C) Representative data from one of three independent experiments. (G–I) Representative plots and statistics from one of three independent experiments. (J and K) Representative plots and statistics from one of five independent experiments; n = 3 in H and I, n = 5 in K. Each symbol represents one sample; data represent mean ± SEM, two-way ANOVA in H and I, two-tailed unpaired t test in K; *** <0.001, **** <0.0001. Source data are available for this figure: SourceData F2.

VMP1 promotes Ca ²⁺ transport across membranes. (A) C-terminal amino acid sequence of VMP1. Lysine residues in the Golgi-to-ER retrieve motif are labeled in blue and the mutated alanine was labeled in red. (B) The interactions between RFP, VMP1, or VMP1-K404/406A with COPI were examined by coimmunoprecipitation. Irrelevant lanes were removed and a dash line is shown. (C) Subcellular localization of FLAG-tagged (N-terminal) VMP1 and VMP1-K404/406A was examined by immunofluorescence. Plasma membrane was labeled by WGA. (D) Putative topology of VMP1 on ER. (E) Putative topology of VMP1-K404/406A on plasma membrane. (F) An illustration of Ca²⁺ transport mediated by VMP1 on ER and VMP1-K404/406A on plasma membrane. (G and H) Ca²⁺ influx in HEK293T cells transfected with empty vector, VMP1, or VMP1-K404/406A was measured by flow cytometry with 8 mM CaCl₂. (I) Ca²⁺ influx in HEK293T or STIM1-KO HEK293T cells transfected with empty vector, VMP1, or VMP1-K404/406A was measured by flow cytometry with 8 mM CaCl₂. (J and K) Whole-cell patch-clamp traces of STIM1-KO HEK293T cells transfected with pCMV-GFP or pCMV-VMP1-K404/406A. Final concentration of 20 mM CaCl₂ was added to bath solution. The holding potential was −100 mV. (B and C) Representative data from one of three independent experiments. (G–I) Representative plots and statistics from one of three independent experiments. (J and K) Representative plots and statistics from one of five independent experiments; n = 3 in H and I, n = 5 in K. Each symbol represents one sample; data represent mean ± SEM, two-way ANOVA in H and I, two-tailed unpaired t test in K; *** <0.001, **** <0.0001. Source data are available for this figure: SourceData F2.

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