FIGURE 4:

Site-directed mutagenesis analyses. (A) Schematic representation of the constructs used for complementation or site-directed mutagenesis experiments. Ndufaf5 was fused to GFP and tandem affinity purification tag (TAP-tag) into the pDV-GFP-CTAP vector, where expression is directed under the control of actin-15 promoter. The V-shaped line represents an intron. M1 is the mutation in the predicted SAM-binding domain (see the text). M2 and M3 are the corresponding mutations to the pathogenic ones in human (M2 equivalent to L159F, and M3 equivalent to L229P). (B) Wild-type and mutant constructs were transfected in ndufaf5⁻ cells. The expressed proteins colocalized with MitoTracker Red in mitochondria to the same extent as depicted in Figure 3A (data not shown). Transfected cells were spread onto SM plates to test the size of the clearing zone after 5 d. The strain transformed with the wild-type protein (ndufaf5⁻ rescue strain) complemented the growth phenotype completely, in contrast to the mutated forms, which showed similar defects as the parental ndufaf5⁻. Bar, 1 cm.