Recombinant Saccharomyces cerevisiae Mitochondrial import inner membrane translocase subunit TIM22 (TIM22)

What is the composition of the TIM22 complex in Saccharomyces cerevisiae?

The TIM22 complex in Saccharomyces cerevisiae consists of seven subunits: Tim22, Tim18, Tim54, Sdh3, Tim9, Tim10, and Tim12. Tim22 is the core translocase subunit responsible for mediating protein insertion into the inner membrane. The three small Tim proteins (Tim9, Tim10, and Tim12) are homologous to each other and form part of the chaperone system. Tim54 likely interacts with the Tim9-Tim10-Tim12 module in the complex. Sdh3 is also a component of respiratory complex II (succinate dehydrogenase), while Tim18 is homologous to Sdh4, which is Sdh3's partner in respiratory complex II .

The composition can be summarized in this table from published research:

What is the function of the TIM22 complex in mitochondria?

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. Proteins imported by this pathway include members of the mitochondrial carrier family, such as the ADP/ATP carrier (AAC), which contains six membrane-spanning regions. These proteins lack cleavable N-terminal targeting sequences but contain internal targeting information distributed throughout the polypeptide chain .

The complex works in coordination with the TOM (Translocase of the Outer Membrane) complex to ensure proper translocation of substrates across both mitochondrial membranes. The TIM22 pathway likely imports all mitochondrial metabolite carriers (about three dozen in yeast) as well as many other integral proteins of the mitochondrial inner membrane, including import components Tim22 and Tim23 .

How can researchers isolate and purify the TIM22 complex for structural studies?

To isolate the endogenous TIM22 complex for structural studies such as cryo-EM, researchers can use the following methodology:

• Genomic tagging: Add a C-terminal twin-strep tag to Tim18 in the yeast genome to facilitate purification .

• Mitochondrial isolation: Isolate mitochondria using a standard protocol such as that described by Berthold et al. (1995) .

• Solubilization: Solubilize mitochondrial membranes using either digitonin (to preserve the TIM22 complex integrity) or Triton X-100 (which disrupts the Tim22·54 complex) .

• Affinity purification: Use the twin-strep tag for affinity purification of the intact complex.

• Size exclusion chromatography: Perform gel filtration to separate the ~300 kDa TIM22 complex from the ~70 kDa Tim9-Tim10 complex .

This approach has successfully been used to determine the structure of the TIM22 complex at a resolution of 3.8 Å .

What methods can be used to study protein-protein interactions within the TIM22 complex?

Several complementary approaches can be used to investigate protein-protein interactions within the TIM22 complex:

• Crosslinking-Mass Spectrometry (XL-MS): This approach has been successfully applied to determine the molecular arrangement of subunits in the human TIM22 complex. Using BS3 crosslinker on the isolated complex generates crosslinks across most components, including the small TIM chaperone complex .

• Co-immunoprecipitation: As demonstrated in studies with Tim9, Tim10, and Tim12, immunoprecipitation with antibodies against one component (e.g., Tim10) can identify interacting partners. For example, when mitochondria from 35S-labeled yeast cells are solubilized with Triton X-100 and immunoprecipitated with anti-Tim10 antibodies, Tim9 and Tim12 co-precipitate, indicating their physical interaction .

• Genetic interaction studies: Suppressor mutations, such as the tim9 199A→T (Tim9 S67C) mutation that suppresses temperature-sensitive phenotypes of tim10-1 and tim12-1 mutants, can reveal functional interactions between components .

• Blue native gel electrophoresis: This technique can separate intact protein complexes and identify subcomplexes, helping to determine the organization of the TIM22 complex components.

How does the organization of small Tim proteins contribute to TIM22 complex function?

The small Tim proteins (Tim9, Tim10, and Tim12) are organized into two distinct hetero-oligomeric assemblies with specific functions in the TIM22 import pathway:

• TIM9·10 complex: This ~70 kDa soluble complex in the intermembrane space consists of approximately 3-4 molecules of Tim9 and 2-3 molecules of Tim10. It is 3-4 fold more abundant than the TIM9·10·12 complex and mediates partial translocation of mitochondrial carrier proteins across the outer membrane .

• TIM9·10·12 complex: This complex contains Tim9, Tim10, and Tim12, possibly consisting of 3-4 subunits of Tim9, two Tim10, and one Tim12 molecule. It is tightly associated with Tim22 in the inner membrane and assists further translocation into the inner membrane in association with TIM22·54 .

The structural arrangement revealed by cryo-EM shows that the small Tim subunits in TIM22 form a hexameric ring that sits on the membrane with a ~45° tilt. One Tim10 subunit is replaced by Tim12, which has an additional C-terminal helix that intercalates between the N- and C-terminal helices of one Tim9 subunit .

Research indicates that the essential role of the small Tim proteins may be at the inner membrane rather than in the intermembrane space. This is supported by genetic studies showing that a Tim9 S67C mutation can restore growth to temperature-sensitive tim10-1 and tim12-1 strains .

What is the substrate specificity of the TIM22 complex?

The TIM22 complex imports a specific subset of mitochondrial inner membrane proteins. A chemical-genetic approach using a small molecule inhibitor called MitoBloCK-1 has helped elucidate the substrate specificity:

• Carrier proteins: The ADP/ATP carrier (AAC) and phosphate carrier are primary substrates of the TIM22 pathway .

• Tim22: The Tim22 protein itself is imported via the TIM22 pathway, indicating a self-assembly mechanism .

• Tafazzin: This phospholipid transacylase, associated with Barth syndrome when mutated, is imported via the TIM22 pathway .

• Tom40: Components of the TOM complex such as Tom40 are also TIM22 substrates .

• Non-substrates: Tim23, a component of the TIM23 complex, is not imported via the TIM22 pathway but rather through a different mechanism .

This substrate specificity appears to be conserved in mammalian mitochondria, as MitoBloCK-1 inhibits the import of the ADP/ATP carrier but not TIM23 substrates in mammalian cells .

How do charged residues in Tim22 contribute to protein translocation?

Structural studies have revealed unusual electrostatic features in Tim22 that likely play critical roles in protein translocation:

• The concave surface of Tim22 has a large negatively charged patch exposed to the membrane on the intermembrane space (IMS) side.

• The transmembrane distance in Tim22 is relatively short due to a TM2-TM3 split on the matrix side.

• Tim22 contains a completely conserved glutamic acid (E140) in TM2 next to a disulfide bond, as well as a conserved aspartic acid (D190) in TM4.

• The TM2-TM3 split is bordered by two lysine residues pointing to each other: a conserved lysine (K127) of TM2 and a lysine in the loop between TMs 3 and 4 (K169).

Mutagenesis studies have demonstrated the critical importance of these charged residues:

• Single mutations E140A and K127A greatly impair yeast cell growth

• Double mutants E140A/D190A and K127A/K169A are essentially lethal to yeast

• The quadruple mutant shows complete growth failure

These charged residues may function by decreasing the thickness of the local lipid membrane, potentially reducing the energy barrier for transmembrane insertion of substrate proteins .

What is the functional relationship between the TIM22 complex and protein quality control systems in mitochondria?

Recent research has uncovered a novel genetic connection between the TIM22 complex and the YME1 protein quality control machinery in Saccharomyces cerevisiae:

• Impairment in the TIM22 complex rescues the respiratory growth defects of cells lacking Yme1, a mitochondrial inner membrane metalloprotease with chaperone-like and proteolytic activities.

• Yme1 is essential for the stability of the TIM22 complex and regulates the proteostasis of TIM22 pathway substrates.

• Compromising the TIM22 complex suppresses the mitochondrial structural and functional defects of Yme1-devoid cells.

These findings suggest that excessive levels of TIM22 pathway substrates could be one reason for respiratory growth defects in cells lacking Yme1. By compromising the TIM22 complex, the imbalance in mitochondrial proteostasis caused by the loss of Yme1 can be compensated for .

How does the structure and function of the TIM22 complex differ between yeast and humans?

While the TIM22 complex is functionally conserved across species, there are significant structural differences between yeast and human versions:

• Composition: The human TIM22 complex includes a novel metazoan-specific subunit called Tim29 (C19orf52) that is not present in yeast. Tim29 is integrated into the mitochondrial inner membrane with its C-terminus exposed to the intermembrane space .

• Function of additional components: Tim29 in humans is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Additionally, Tim29 contacts the TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space .

• Functional implications: The presence of Tim29 suggests that the mitochondrial import machinery has evolved additional components and mechanisms in higher organisms, potentially to accommodate increased complexity or regulatory requirements.

What techniques can be used to study the real-time dynamics of protein import via the TIM22 complex?

To investigate the real-time dynamics of protein import via the TIM22 pathway, researchers can employ several approaches:

• In vitro import assays: Using radiolabeled precursor proteins and isolated mitochondria, researchers can monitor the time-dependent accumulation of imported proteins. This approach has been used to demonstrate that import of AAC is inhibited in tim10-1 mutant mitochondria compared to wild-type .

• Chemical-genetic approaches: Small molecule inhibitors like MitoBloCK-1, which was identified through a synthetic lethal screen with a tim10-1 mutant, can be used to temporally control TIM22 pathway function and observe the consequences on protein import .

• Crosslinking-immunoprecipitation assays: By using chemical crosslinkers at different time points during import, researchers can capture and identify transient interactions between substrates and TIM22 components .

• Arrested translocation intermediates: Creating conditions where translocation is arrested at specific stages allows researchers to characterize discrete steps in the import pathway. For example, a translocation intermediate bound to Tim9 S67C can be localized to the intermembrane space, associated with the inner membrane .

How can recombinant TIM22 proteins be efficiently produced for structural and functional studies?

Efficient production of recombinant TIM22 components for research is challenging due to their hydrophobic nature and complex assembly. Here is a methodological approach based on successful recombinant protein production strategies in S. cerevisiae:

• Selection of expression host: S. cerevisiae is an excellent host for TIM22 components due to its well-established genetics and native mitochondrial environment. For production benchmarks in S. cerevisiae, see the comparison table below:

• Vector design: For TIM22 expression, consider:

  • Using strong but tunable promoters like GAL1 or ADH1

  • Including appropriate mitochondrial targeting signals if expressing individual components

  • Adding affinity tags (His, Strep, FLAG) for purification, positioned to avoid interference with folding

• Codon optimization: Optimize codons for expression in S. cerevisiae, especially for non-yeast proteins.

• Strain engineering: Consider using strains with:

  • Reduced protease activity (pep4Δ, prb1Δ)

  • Enhanced protein folding capacity (upregulated chaperones)

  • Deletion of genes for multidrug resistance pumps (PDR5, SNQ2) if using selection markers

• Induction and growth conditions: Carefully control temperature, pH, and carbon source. For galactose-inducible systems, pre-grow cells in raffinose to avoid glucose repression, then induce with galactose.

• Verification of expression: Confirm correct expression and localization using methods such as:

  • Western blot analysis of mitochondrial fractions

  • Blue native PAGE to check complex assembly

  • Functional complementation of temperature-sensitive mutants

These approaches can help overcome the challenges associated with producing functional TIM22 complex components for research purposes.

What experimental approaches can be used to investigate the mechanism of membrane insertion by the TIM22 complex?

Investigating the precise mechanism of membrane insertion by the TIM22 complex requires a multifaceted approach:

• Site-specific crosslinking: Using amber suppression technology to incorporate photoreactive amino acids at specific positions in TIM22 substrates can help map the path of the substrate through the complex during translocation.

• Membrane potential manipulation: Since TIM22-mediated insertion is dependent on membrane potential, controlled manipulation of the membrane potential can help dissect the energetic requirements of different steps in the insertion process.

• Single-molecule techniques: Approaches such as optical tweezers or atomic force microscopy could potentially be adapted to measure the forces involved in membrane insertion.

• Reconstitution experiments: Purified TIM22 complex components can be reconstituted into liposomes to study the minimal requirements for membrane insertion in a defined environment.

• Structural studies of insertion intermediates: Cryo-EM studies of the TIM22 complex with stalled substrate insertion intermediates could provide snapshots of the insertion process.

Current structural data does not reveal an obvious translocation channel for substrate import, which is surprising given previous suggestions that the TIM22 complex might function as a twin-pore translocase. Two possible models have been proposed:

a) The observed structure may represent an idle state, and the TIM22 complex (particularly Tim22) may undergo conformational changes to oligomerize and form pores when activated by membrane potential.

b) A single Tim22 subunit may be sufficient to act as a transmembrane insertase, similar to the bacterial insertase YidC, potentially using charged residues to create a hydrophilic environment that facilitates membrane insertion .