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The presequence-binding domain of Tim50 is essential. (A) Diploid yeast cells carrying a single chromosomal deletion of TIM50366-476 were sporulated and subjected to tetrad dissection. (B) Yeast cells containing a chromosomal deletion of TIM50, complemented by a TIM50-containing plasmid carrying URA3, were transformed with plasmid constructs encoding the indicated Tim50 versions and subjected to plasmid loss on 5-FOA–containing medium. (C) Western blot analysis of mitochondria isolated from the cells described in A with the indicated antibodies. (D) In organello photo cross-linking of presequence probes using mitochondria from indicated strains for 30 min. Samples were analyzed as in C.