Fig. 1. In vivo and in organello analysis of the CL dependence of the Tim23-Tim50 interaction.

(A) Coimmunoprecipitation analysis of the Tim23-Tim50 interaction. Digitonin lysates of mitochondria from yeast strains containing full-length Tim50 (left) or Tim50^IMS (right) were immunoprecipitated with affinity-purified antibodies to Tim23 (αTim23) or Tim50 (αTim50) or preimmune immunoglobulins (PI). Unbound (Sup.) and bound (Pellets) fractions were analyzed by SDS-PAGE and immunodecoration with antibodies against Tim50, Tim23, or Tim17, as indicated. Total and Sup. lanes indicate 20% of starting material; all other lanes represent undiluted samples. (B) Synthetic lethality of tim23yl and Tim50^IMS. Yeast cells containing a chromosomal deletion of TIM23 complemented with a TIM23-containing URA3 plasmid were generated in a Tim50 or Tim50^IMS background (delineated by dotted boxes). Cells were transformed with centromeric plasmids carrying either Tim23 or tim23yl, as indicated, and subjected to URA3 plasmid loss by plating on glucose-containing medium with 5-fluoroorotic acid (5-FOA). Cells were incubated at 30°C (upper plate) or 24°C (lower plate). (C) Genetic interaction of Δcrd1 and tim23yl. Tenfold serial dilutions were prepared from yeast cells containing WT Tim23 or the tim23yl mutant in a background of normal CL synthesis or CL synthase knockouts (Δcrd1). Cells were spotted onto YPD or YPLac medium as shown and grown at the indicated temperatures.