Figure 3. Homotypic interactions of... | Rockefeller University Press

Figure 3.

$Figure 3. Homotypic interactions of MFN1. (A) The sizes of wild-type (wt) MFN1-MGD (theoretical molecular mass, 49.5 kD) and the K88A mutant (both at 0.4 mM) were determined by analytical ultracentrifugation in the presence of 2 mM indicated nucleotides. The estimated molecular masses are given above the peaks (in kilodaltons). The data are representative of at least three repetitions. (B) Purified (P) and reconstituted dmATL TM-containing MGD (domain structure shown above) was subjected to floatation analysis. Top (T) and bottom (B) fractions were analyzed by SDS-PAGE and Coomassie staining. M, molecular marker shown in kilodaltons. (C) Membrane tethering by MGD-TMATL followed by a visual assay. Proteoliposomes containing MFN1 (protein/lipid ratio 1:2,000) and rhodamine-labeled lipids were analyzed by confocal microscopy. One aliquot was sampled immediately, and a second was taken after incubation at 37°C with 10 mM of the indicated nucleotide for 30 min. The data are representative of at least three repetitions. Bar, 50 µm. (D) As in C, but measured by absorbance at 405 nm. (E) HA-tagged and Flag-tagged full-length (FL) human MFN1 were cotransfected into HEK293T cells and solubilized in digitonin or transfected individually followed by mixing of the digitonin-solubilized cell extracts. Immunoprecipitation (IP) was performed with anti-HA or anti-Flag agarose beads. When indicated, 1 mM nucleotides was added and incubated. The samples were analyzed by SDS-PAGE and immunoblotting (IB). 10% of the starting material (load) and the material not bound to the antibodies (unbound) was also analyzed.$

Homotypic interactions of MFN1. (A) The sizes of wild-type (wt) MFN1-MGD (theoretical molecular mass, 49.5 kD) and the K88A mutant (both at 0.4 mM) were determined by analytical ultracentrifugation in the presence of 2 mM indicated nucleotides. The estimated molecular masses are given above the peaks (in kilodaltons). The data are representative of at least three repetitions. (B) Purified (P) and reconstituted dmATL TM-containing MGD (domain structure shown above) was subjected to floatation analysis. Top (T) and bottom (B) fractions were analyzed by SDS-PAGE and Coomassie staining. M, molecular marker shown in kilodaltons. (C) Membrane tethering by MGD-TM^ATL followed by a visual assay. Proteoliposomes containing MFN1 (protein/lipid ratio 1:2,000) and rhodamine-labeled lipids were analyzed by confocal microscopy. One aliquot was sampled immediately, and a second was taken after incubation at 37°C with 10 mM of the indicated nucleotide for 30 min. The data are representative of at least three repetitions. Bar, 50 µm. (D) As in C, but measured by absorbance at 405 nm. (E) HA-tagged and Flag-tagged full-length (FL) human MFN1 were cotransfected into HEK293T cells and solubilized in digitonin or transfected individually followed by mixing of the digitonin-solubilized cell extracts. Immunoprecipitation (IP) was performed with anti-HA or anti-Flag agarose beads. When indicated, 1 mM nucleotides was added and incubated. The samples were analyzed by SDS-PAGE and immunoblotting (IB). 10% of the starting material (load) and the material not bound to the antibodies (unbound) was also analyzed.