(A) Diagram of Tim44 showing functional regions and interaction partners of the NTD. Residues from 156 to 184 are indicated in yellow. Residues crosslinked to Tim23 in our previous report (Ting et al., 2014) are in red; residue G173, the suppressor reported here, and R180, the previously isolated ts mutant (Schiller et al., 2008), which affects Tim23 and mtHsp70 association, are in green and blue, respectively. The N terminal segment of Tim44 (residues 51 to 82) implicated in interaction with Pam16 is indicated (Schilke et al., 2012). The mutant D345A/E350A and residues D165Bpa, S271Bpa, A391Bpa used for crosslinking in combination the G173V suppressing substitution are indicated in orange and dark red, respectively. (B) Growth phenotype. Ten-fold serial dilutions of tim44-Δ cells expressing Tim44 variants were plated on minimal media and incubated at the indicated temperatures and times. For each temperature, all strains were plated on the same plate; strains not relevant to this report were cropped out (indicated by white space). (C) Co-immunoprecipitation with Tim23. Mitochondria were solubilized by treatment with digitonin. Solubilized material was subjected to immunoprecipitation using Tim23-speciﬁc antibodies crosslinked to protein A beads. Precipitates were analyzed by SDS-PAGE and immunoblotting using antibodies speciﬁc for the indicated proteins. 10% of input for co-immunoprecipitation was used as a loading control. Signals were quantified by Image J software (RRID: SCR_003070) and plotted as percentages of the wt control reaction value. Data represent the mean ± standard deviation, with n = 3. The data of the figure can also be seen in Figure 5—source data 1. (D) 35S-labeled precursor of cytochrome b2-(167)Δ19-DHFR was imported into wild-type (WT), tim44D345AK, and tim44D345AK/G173V mitochondria at 25°C. Where indicated, mitochondria were subsequently treated with proteinase K (Prot. K). Samples were analyzed by SDS-PAGE and autoradiography. p, precursor; I, intermediate; m, mature. (E) Growth phenotype. Ten-fold serial dilutions of tim44-Δ cells expressing the indicated Tim44 variants were plated on minimal media and incubated at the temperatures and for the times indicated. (F) Yeast strains expressing tim44D345A/E350A with Bpa incorporated at position D165, S271 or A391 were subjected to UV irradiation (+), or as a control not exposed (-). Tim44 was then affinity purified via its N-terminal His6 tag, followed by SDS-PAGE and immunoblotting with the indicated antibodies. Migration of size standards, in kDa, are indicated (left); Tim44 (44NTD or 44CTD), Tim23 (23), and Tim17 (17) reactive bands are indicated. The asterisk indicates non-specific cross-reactive bands.