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Figure 4


Fig. 4. Effect of MIB-FLAG constructs carrying mutation in the GGVG-motif on mitochondrial morphology. (A) MIB-FLAG proteins with the indicated mutations in the GGVG-motif were expressed in HeLa cells and examined by fluorescence microscopy as in Fig. 3. The constructs V13A and V190A carrying a mutation in the arbitrarily selected tetrapeptide region (containing GV) outside the GGVG-motif were examined as the negative controls. Bars, 10 µm. (B) Cells with the indicated mitochondrial morphology shown in A were quantified. More than 100 cells were counted in at least three distinct optical fields. Control: empty vector. (C) Expression levels of the indicated constructs were analyzed by SDS-PAGE and subsequent immunoblotting using anti-flag IgG. (D) Wild-type MIB-FLAG or the GGAG-mutant were exogenously expressed in HeLa cells, and the cytosolic fractions incubated with MBP- or MBP-Mfn1{Delta}C-conjugated beads. Proteins bound to the beads were eluted by buffer containing the indicated concentrations of NaCl, and eluted proteins were analyzed by SDS-PAGE and subsequent immunoblotting with anti-flag antibodies.





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