Figure 1. The function of Tim44 can be rescued by its two domains expressed in trans but not by either of the domains alone.

(A) Schematic representation of Tim44 domain structure (numbering according to yeast Tim44 sequence). pre. - presequence (B and C) A haploid yeast deletion strain of TIM44 carrying the wild-type copy of TIM44 on a URA plasmid was transformed with centromeric plasmids carrying indicated constructs of Tim44 under control of endogenous promoter and 3'UTR. Cells were plated on medium containing 5-fluoroorotic acid and incubated at 30°C. The plasmid carrying wild-type Tim44 and an empty plasmid were used as positive and negative controls, respectively. (D) Total cell extracts of wild-type yeast cells transformed with plasmids coding for indicated Tim44 constructs under GPD promoter were analysed by SDS–PAGE and immunoblotting against depicted antibodies. *, ** and *** - protein bands detected with antibodies raised against full-length Tim44.

DOI: http://dx.doi.org/10.7554/eLife.11897.003

Figure 1—figure supplement 1. Two domains of Tim44 do not interact stably with each other.

(A) Purified His₆-Tim44(43–263) was incubated with purified Tim44(211–431) either in low-salt (20 mM Tris/HCl, 50 mM NaCl, 10 mM imidazole, pH 8.0) or high-salt buffer (20 mM Tris/HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0) for 5 min at 25°C. The NiNTA-agarose beads were added and the mixture gently rolled for 30 min at 4°C. After three washing steps with the same buffer, bound proteins were eluted with the buffer containing 300 mM imidazole. Total (T, 10%), flow-through (FT, 10%), and bound (B, 100%) fractions were analyzed by SDS–PAGE followed by Coomassie staining. (B) Mitochondria were isolated from yeast cells in which the function of the full-length Tim44 was rescued by coexpression of N- and C-terminal domains separately (N+C). In His₉N+C mitochondria, the N-terminal domain contained an additional His₉ tag. Mitochondria were solubilized with digitonin-containing buffer and incubated with NiNTA-agarose beads at 4°C. After three washing steps, proteins specifically bound to the beads were eluted with Laemmli buffer containing 300 mM imidazole. Total (T, 10%), flow-through (FT, 10%), and bound (B, 100%) fractions were analyzed by SDS–PAGE followed by imunoblotting using antibodies to Tim44.

DOI: http://dx.doi.org/10.7554/eLife.11897.004