Domain organization of Pam16 and Pam18. (A) Schematic representation of Pam18 and Pam16 with amino acids corresponding to predicted domains indicated. Pam18: aa 1-60, intermembrane space (IMS); aa 65-83, transmembrane (TM); aa 101-168, J-domain (J). Pam16: aa 1-29, hydrophobic (H); aa 54-117, J-like domain (J). Amino-acid alterations analyzed in this report are indicated by asterisks or, in the N terminus of Pam16, by a dotted line, indicating K19, E23, R26, and Q27. The segment of Pam18 and Pam16 used in structural determination (Mokranjac et al., 2006) is indicated by the solid line. (B) Interaction of Pam18 and Pam16 J-domians (Mokranjac et al., 2006). A symmetrical set of interactions were observed between Pam16 and Pam18 in a strongly interacting hydrophobic pocket that includes: L150 of Pam18 and L97 of Pam16, indicated. The conserved HPD found in J-domains and required for stimulation of Hsp70 ATPase activity is also indicated.

Analysis of mutations affecting stability of the Pam16:Pam18 heterodimer. (A) Growth phenotypes. Tenfold serial dilutions of wt, pam18_L150W, and pam16_L97W cells were spotted onto rich glucose-based media, followed by incubation at the indicated temperatures for 2 d. (B) Interaction of purified Pam16 and Pam18. Purified proteins (3.1 μM) were incubated together for 30 min at 23°C: wt Pam18 (18), wt Pam16 (16), Pam16_L97W (16_L/W), or Pam18_L150W, (18_L/W). CoIPs was carried out using Pam18- and Pam16-specific antibodies. The samples were analyzed by SDS-PAGE and stained with Coomassie Blue. Samples containing 100% of the input served as a control for immunoprecipitation efficiency (bottom). Controls (C) were Pam18 (C₁), Pam18_L150W (C₂), Pam16 (C₃), and Pam16_L97W (C₄). (C and D) In organellar analysis of Pam18 and Pam16. wt, pam18_L150W (18_LW), or pam16_L97W (16_LW). (C) Mitochondria were incubated in 1% Triton X-100 and then subjected to immunoprecipitation by using Pam18- and Pam16-specific antibodies, as indicated by brackets, followed by SDS-PAGE and immunoblotting by using Pam18 and Pam-specific antibodies. Fifty percent of soluble material after lysis was used as a loading control (50% input). (D) Coimmunoprecipitation of the Pam18:Pam16 complex with the core Tim23 translocon complex. Mitochondria were solubilized with buffer containing 1% digitonin, and the supernatants were subjected to immunoprecipitation using Tim23-specific antibodies. The samples were analyzed by SDS-PAGE and immunoblotted against Tim23-, Tim50-, Tim44-, Tim17-, Pam16- and Pam18-specific antibodies. Twenty-five percent of total soluble material after lysis was used as a loading control (25% input).

Functional importance of IMS domain of Pam18. (A) Interaction of Pam18_IMS with Tim17. Fusions between GST and Pam18_IMS domains (Pam18 [WT) and Pam18_A57P [A57P], and GST alone as a control) were immobilized on glutathione-agarose beads, and then they were incubated in wt mitochondrial lysates. Bound proteins were subjected to SDS-PAGE and immunoblotted with antibodies specific for the indicated proteins. Untreated glutathione-agarose beads (control) were used as a negative control. (B) Effect of combination of the A57P and L150W alterations in Pam18 on cell growth. Tenfold serial dilutions of Δpam18 cells carrying a plasmid expressing either wt or indicated PAM18 mutant gene were plated on rich glucose-based media and grown at 23°C for 3 d and at 30 or 34°C for 2 d. (C) Expression levels of Pam18 mutant proteins. Whole cell extracts from pam18_A57P, pam18_L150W, and pam18_A57P/L150W were separated by SDS-PAGE and subjected to immunoblot analysis by using antibodies specific for Pam18, and as a loading control Tim23. All samples were run on the same gel, but lanes unrelated to the experiment presented here were removed.

Analysis of the functional importance of N terminus of Pam16 by using Pam16:Pam18 chimeras. (A) Schematic representation of Pam16 deletion construct and Pam16:Pam18 chimeras used. Because Pam16_51-117 lacks a mitochondrial targeting sequence, it was expressed with the cleavable Su9 targeting sequence (pre) at the N terminus as reported previously (Mokranjac et al., 2006). Other abbreviations are as in the legend of Figure 1. (B and C) Tenfold serial dilutions of Δpam16 cells carrying a centromeric plasmid expressing indicated protein were plated on rich media and incubated at the indicated temperatures for 3 d.

Analysis of the functional importance of the N terminus of Pam16 by using full-length protein. (A) Growth phenotypes of Δpam16 cells expressing the indicated N-terminal amino-acid alterations in full-length Pam16 (KERQ indicates K19A/E23G/R26A/Q27A). Tenfold serial dilutions were plated on rich glucose-based medium and grown at 30 or 37°C for 2 d or on rich glycerol-based medium and incubated at 37°C for 5 d. (B) Growth phenotypes of Δpam16 Δpam18 cells expressing wt or the indicated N-terminal amino-acid alterations in full-length Pam16 and either wt Pam18 or Pam18_L150W. KE indicates K19A/E23G. Tenfold serial dilutions were plated on rich glucose-based medium at the indicated temperatures for 3 d. (C) Growth phenotypes of Δpam16 cells expressing either full-length wt Pam16 or Pam16 either with N-terminal amino-acid alterations and/or the L97W alteration in the J-like domain. Tenfold serial dilutions were plated on rich glucose-based medium and incubated at the indicated temperatures for 3 d.

Association of Pam16 having alterations in the N terminus with the translocon. (A) Pam16-Pam18 association. Equivalent amounts of wt and pam16 (16) mutant mitochondria were lysed by incubation with mitochondrial lysis buffer containing 1% Triton X-100. Lysates were subjected to immunoprecipitation by using Pam18-specific antibodies. The samples were analyzed by SDS-PAGE and immunoblotted against antibodies specific for Pam18 and Pam16. Fifty percent of soluble material after lysis was used as a loading control (50% load). pam16_K19A/E23G, 16_KE; pam16_{K19A/E23G/R26A/Q27A}, 16_KERQ. (B and C) Association of Pam18 and Pam16 mutants with the Tim23-core complex. Equivalent amount of mitochondria from cells expressing the indicated combination of Pam16 and/or Pam18 mutant proteins were solubilized by incubation in buffer containing 1% digitonin. Supernatants were subjected to immunoprecipitation using Tim23-specific antibodies. The samples were analyzed by SDS-PAGE and immunoblotted with Tim23-, Tim17-, Tim44-, Pam16-, and Pam18-specific antibodies. Twenty-five percent of total soluble material after lysis was used as a loading control (25% I).

Suppression of defects caused by alterations in the N terminus of Pam16 by alterations in Tim44. (A) Tenfold serial dilutions of Δtim44 Δpam16 cells expressing wt or mutant TIM44 genes and Pam16[1-27]:Pam18 chimeras having the indicated amino acid alterations in the N terminus of the Pam16 segment were spotted on rich glucose medium and incubated at the indicated temperatures for 3 d. Dotted lines separate individual plates, which were all spotted at the same time. (B) Mitochondrial membrane association analysis of chimeric mutant proteins and suppressors. Sonicated mitochondria were subjected to centrifugation and equivalent samples of supernatant (S) and pellet (P) fractions, and unfractionated extract (T), were analyzed by SDS-PAGE and immunoblotted with Pam16-, Pam18-, and Mge1-specific antibodies. (C) Ten-fold serial dilutions of Δtim44 Δpam16 cells expressing wt or mutant TIM44 genes and full-length Pam16 with various amino-acid alterations were spotted on rich glucose medium and incubated at the indicated for 3 d at 23°C or 2 d at 30°C. Dotted lines separate individual plates, which were all spotted at the same time.