The Tim21 binding domain connects the preprotein translocases of both mitochondrial membranes

Abstract

Proteins destined for the mitochondrial matrix are imported by the translocase of the outer membrane--the TOM complex--and the presequence translocase of the inner membrane--the TIM23 complex. At present, there is no structural information on components of the presequence translocase. Tim21, a subunit of the presequence translocase consisting of a membrane anchor and a carboxy-terminal domain exposed to the intermembrane space, directly connects the TOM and TIM23 complexes by binding to the intermembrane space domain of the Tom22 receptor. We crystallized the binding domain of Tim21 of Saccharomyces cerevisiae and determined its structure at 1.6 A resolution. The Tim21 structure represents a new alpha/beta-mixed protein fold with two alpha-helices flanked by an extended eight-stranded beta-sheet. We also identified a core sequence of Tom22 that binds to Tim21. Furthermore, negatively charged amino-acid residues of Tom22 are important for binding to Tim21. Here we suggest a mechanism for the TOM-TIM interaction.

Figure 1

Tim21_IMS interaction with Tom22_IMS. (A) Purified GST–Tom22_IMS and His₆–Tim21_IMS were mixed and applied to Ni-NTA-agarose. After washing, bound proteins were eluted with imidazole and analysed by SDS–polyacrylamide gel electrophoresis and stained with Coomassie R-250. (B) ¹H NMR spectroscopic detection of complex formation between Tim21_IMS and Tom22_IMS. Spectra of Tom22_IMS were recorded in the absence or presence of Tim21_IMS. Grey panels indicate changes in peak intensity. GST, glutathione S-transferase; Tim21_IMS, translocase of the inner membrane; Tom22_IMS, translocase of the outer membrane.

Figure 2

Crystal structure of Tim21_IMS. (A) Views of the molecular structure of Tim21_IMS (top) with labelled α-helices (green) and β-strands (red). Images were generated using Molscript (Kraulis, 1991). Surface presentations of the Tim21_IMS molecule are shown (bottom). Positive and negative potentials are coloured blue and red, respectively. The images were generated using Grasp (Nicholls et al, 1991). (B) Schematic representation of the Tim21_IMS fold. (C) Sequence of Tim21_IMS and distribution of secondary structure elements. (D) Hydrogen bonds (dashed) between the helical part and the antiparallel β-sheet. Tim21_IMS, translocase of the inner membrane.

Figure 3

Surface presentations of Tim21_IMS. Conserved positively charged (blue), negatively charged (red), amphipathic (green) and hydrophobic (black) residues on the surface are shown. Ten unicellular organisms were compared (Saccharomyces cerevisiae, Aspergillus fumigatus, Neurospora crassa, Candida albicans, Kluyveromyces lactis, Aphis gossypii, Candida glabrata, Desulfomusa hansenii, Yarrowia lipolytica, Schizosaccharomyces pombe). Images were generated using DINO (http://www.dino3d.org/). Tim21_IMS, translocase of the inner membrane.

Figure 4

The Tim21_IMS–Tom22_IMS association involves electrostatic interaction. (A) The immobilized IMS domain of Tim21 was incubated with solubilized yeast mitochondria. After washing in the presence of NaCl, bound proteins were eluted and analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting with Tom40 antibodies. (B) Sequence of Tom22_IMS and synthetic peptides. (C) Isolated Tim21_IMS was incubated with purified Tom22_IMS (P1 recom.) or the indicated peptides and crosslinked with glutaraldehyde. Samples were analysed by SDS–PAGE and Coomassie staining. Tim21_IMS, translocase of the inner membrane; Tom22_IMS, translocase of the outer membrane.