Structure of TimA. (A) Representation of the domain organization in the Tim44 family of proteins. Black denotes the inner membrane targeting sequence for mitochondria (humans, Saccharomyces, Schizosaccharomyces) or bacteria (Azorhizobium, Caulobacter). (B) Model of TimA (CC3741) from C. crescentus based on the structures of human and yeast Tim44, represented as cartoon and a solvent-excluded surface. Hydrophobic residues are cyan and other residues are red or green. Aromatic residues (F¹⁰⁴, F¹⁰⁹, Y¹¹⁷, Y¹²⁴, F¹⁴⁴, F¹⁶², W²¹¹, F²¹³, W²²⁴, and F²²⁸) are shown in stick representation. The structure was assessed with the Prosa2003, ProQ, and Verify3D quality scores (see Fig. S1).

Location and topology of TimA and TimB. (A) Domain structure of Tim14 (41, 42) and TimB. (Black, signal sequence; TM, transmembrane domain.) The “J-domain” (31) that interacts with Hsp70 is shown (detailed sequence analysis provided in Fig. S2). (B) Membranes were fractionated on sucrose gradient and analyzed by SDS/PAGE and immunoblotting. (C) Inner membrane vesicles (IM) were extracted with alkali and the pellet (P) and supernatant (S) fractions analyzed by Coomassie staining (Upper) and immunoblots for TimA, TimB, the integral membrane protein DivJ, and peripheral membrane protein F₁β. (D) Fluorescence microscopy of the OmpA-mCherry strain shows the periplasmic location of the protein. (E) Caulobacter cells were incubated without (−) or with (+) polymyxin B and proteinase K as indicated and then analyzed by SDS/PAGE and immunoblotting.

TimA and TimB do not associate in a membrane complex. (A) Inner membrane vesicles (IM) solubilized with DDM were subject to immunoprecipitation assays with pre-immune serum (−) or antiserum recognizing TimA (A) or TimB (B). The immuno-depleted supernatants and the precipitates were analyzed by SDS/PAGE and immunoblotting with the sera as shown. (B) Immunoprecipitations from inner membrane vesicles (1 mg total protein) using either pre-immune serum (pre-) or immune serum raised to TimA were analyzed by SDS/PAGE and Coomassie staining, with the identity of the precipitated proteins determined by MS. (C) Inner membrane vesicles were solubilized with the detergent DDM and analyzed by blue native PAGE and immunoblotting for TimA and TimB. The Coomassie blue–stained gel (stain) is also shown.

TimB can be converted to function in mitochondrial protein import. (A) Model of PbTimB-A¹³⁹N (blue) binding to Tim16 (orange) based on the published Tim14-Tim16 interaction (2GUZ). In Tim14, the asparagine residue (green) forms a pair of hydrogen bonds with an asparagine in Tim16. In CcTimB and PbTimB, the native residue at this position is an alanine (red). (B) TimB from C. crescentus and P. bermudensis were engineered for expression in yeast by adding an N-terminal mitochondrial targeting sequence and transmembrane domain. A point mutation was introduced at residue 139 of PbTimB (TimB-A¹³⁹N) to replace the alanine with an asparagine residue. After transformation with plasmids carrying the engineered TimB constructs, Tim14/Δtim14 yeast cells were sporulated. Tetrads were dissected: 2 spores of tetrads 1 and 2 formed viable colonies; 4 spores of tetrads 3 and 4 formed viable colonies. Arrows indicate subculturing the cells from tetrad 4 to measure growth phenotype, verifying that the 2 smaller colonies are Δtim14 cells kept viable by PbTimB(A¹³⁹N).