Recombinant Rat Mitochondrial import inner membrane translocase subunit Tim22 (Timm22)

What is the functional role of Tim22 in mitochondrial protein translocation?

Tim22 serves as the core channel-forming subunit of the TIM22 complex, which mediates the insertion of multi-transmembrane domain proteins with internal targeting signals into the mitochondrial inner membrane. It forms a hydrophilic, high-conductance channel that is voltage-activated and specifically responds to internal targeting signals, but not to presequences . The TIM22 pathway is essential for importing metabolite carriers and membrane-embedded components of the mitochondrial import machinery, functions that are critical for cellular survival . Mechanistically, Tim22 combines three essential functions in a single protein component: recognition of targeting signals, formation of the translocation channel, and transduction of energy required for the import process .

How is the TIM22 complex organized and what are its key components?

The TIM22 complex is a sophisticated multisubunit molecular machine comprising several key components:

• The 300-kDa insertion complex in the inner membrane consists of Tim22, Tim18, Tim54, Tim12, and a fraction of Tim9 and Tim10 .

• The small Tim protein chaperone complexes in the intermembrane space: Tim9-Tim10 and Tim8-Tim13 complexes, which form 70-kDa hexameric structures .

• Tim22 serves as the central channel-forming component .

The small Tim proteins function as chaperones that maintain hydrophobic membrane proteins in an import-competent state as they traverse the aqueous intermembrane space . They escort substrates to the insertion complex, which then mediates the actual insertion of proteins into the membrane . The quaternary structure of the TIM22 complex appears to be stabilized by intramolecular disulfide bonds in Tim22, which influence its interaction with other subunits like Tim18 and Tim54 .

What types of substrates are imported via the TIM22 pathway?

The TIM22 pathway specifically handles:

• Hydrophobic multi-transmembrane spanning proteins with internal targeting signals .

• Metabolite carriers such as the ADP/ATP carrier (AAC), phosphate carrier (PIC), and dicarboxylate carrier (DIC) .

• Membrane-embedded components of the mitochondrial import machinery .

These proteins typically lack N-terminal presequences and instead contain internal targeting signals that are recognized by receptor proteins on the translocase of the outer membrane (TOM complex) . Upon translocation through the TOM complex, these precursor proteins are bound by the small Tim chaperone complexes that maintain them in an import-competent state by preventing aggregation in the aqueous intermembrane space .

How do the intramolecular disulfide bonds in Tim22 affect the stability and function of the TIM22 complex?

The intramolecular disulfide bonds in Tim22, formed between conserved cysteine residues (particularly C42 and C141), play a crucial role in maintaining the structural integrity of the TIM22 complex . Research has shown that:

• Mutation of these conserved cysteines (C42S, C141S, or C42/141S) destabilizes the TIM22 complex, resulting in slightly smaller apparent sizes of the complex as detected by blue-native PAGE .

• The absence of the disulfide bond alters the quaternary structure of the TIM22 complex, facilitating the exchange between newly imported Tim22 and pre-existing Tim22 in the complex .

• Wild-type Tim22 with intact disulfide bonds shows a higher affinity for other subunits of the TIM22 complex (Tim54 and Tim18) compared to Cys→Ser mutants lacking the disulfide bond .

• While Tim22 Cys→Ser mutant cells grow normally under standard conditions, they show compromised growth when carrier proteins (AAC, PIC, or DIC) are overexpressed, indicating that the disulfide bond becomes functionally critical when the TIM22 complex must handle excess substrate proteins .

These findings suggest that Tim22 spontaneously forms a disulfide bond between the conserved Cys residues after proper folding, which is achieved by correct assembly into the TIM22 complex that brings the Cys residues into close proximity .

What experimental approaches can be used to study Tim22 channel activity?

Studying Tim22 channel activity requires specialized techniques:

• Reconstitution of Purified Tim22 in Liposomes: This approach involves isolating Tim22 and reconstituting it in artificial membranes to study its channel properties in isolation .

• Electrophysiological Measurements: These can demonstrate that reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters. This technique also reveals that the channel is voltage-activated and specifically responds to internal targeting signals .

• Substrate Response Assays: These assess how the Tim22 channel responds to different substrates, showing that it specifically recognizes internal targeting signals but not presequences .

• Voltage-Gating Analysis: This approach examines how membrane potential affects Tim22 channel activity, providing insights into the energy transduction mechanism .

When conducting these experiments, it's essential to include appropriate controls such as channels formed by other mitochondrial translocases (like TIM23) to distinguish Tim22-specific activities.

How does the chemical crosslinking-mass spectrometry (XL-MS) approach contribute to understanding the TIM22 complex architecture?

The XL-MS approach has proven valuable for determining the molecular arrangement of subunits within the human TIM22 complex :

• Methodology: The technique involves crosslinking the isolated TIM22 complex using crosslinkers like BS3, followed by mass spectrometry analysis to identify which proteins are in close proximity to each other .

• Comprehensive Subunit Mapping: XL-MS generates crosslinks across the majority of TIM22 components, including the small TIM chaperone complex, providing a detailed map of protein-protein interactions within the complex .

• Unexpected Features: The crosslinking data has uncovered several unexpected features of the TIM22 complex architecture that were not predicted by sequence analysis or homology modeling .

• New Research Avenues: These unexpected findings have opened new avenues for deeper investigation into the steps required for TIM22-mediated translocation in humans, particularly the differences between human and yeast systems .

This approach is particularly valuable because it provides structural information about protein complexes in their native state, complementing other structural biology techniques like X-ray crystallography or cryo-EM.

What strategies can be employed to identify small molecule modulators of the TIM22 pathway?

Identifying small molecule modulators of the TIM22 pathway can be achieved through chemical-genetic screening approaches:

• Synthetic Lethal Screening: Using temperature-sensitive mutants (e.g., tim10-1) to screen for small molecules that cause synthetic lethality at permissive temperatures. This approach identifies compounds that might target mutant proteins or other components of the TIM22 pathway .

• Strain Engineering: Generating suitable screening strains by removing genes for multidrug resistance pumps to increase sensitivity to small molecules .

• Cross-Validation: Confirming that identified compounds specifically affect the TIM22 pathway by testing their effects on different mitochondrial import pathways and various substrate proteins .

• Mechanistic Studies: Using the identified compounds as tools to elucidate details of protein translocation mechanisms, particularly substrate specificity of the small Tim proteins and their binding mechanisms .

This chemical-genetic approach provides a powerful method to develop new tools for mechanistic studies in protein translocation and may be particularly useful for characterizing protein translocation in mammalian mitochondria, where genetic tools are more limited .

What in vitro assays can be used to evaluate Tim22 assembly into the TIM22 complex?

Several in vitro assays can assess Tim22 assembly into the TIM22 complex:

• Radiolabeled Protein Import Assays: Using radiolabeled wild-type Tim22 to track its import and assembly into the TIM22 complex in isolated mitochondria .

• Blue-Native PAGE (BN-PAGE): After solubilizing mitochondria with detergents like digitonin (1%), BN-PAGE can be used to analyze the 300-kDa TIM22 complex and detect differences in complex size and stability between wild-type and mutant variants .

• Co-Immunoprecipitation: This technique can reveal interactions between Tim22 and other components of the TIM22 complex (Tim18, Tim54), helping to determine how structural features like disulfide bonds affect these interactions .

• Heat Treatment Stability Assays: Exposing mitochondria to elevated temperatures (e.g., 37°C) before analysis can reveal differences in complex stability between wild-type and mutant variants .

These assays have revealed, for example, that assembly of wild-type Tim22 into the TIM22 complex is accelerated in mitochondria containing Tim22 Cys→Ser mutations, suggesting that the lack of disulfide bonds alters the quaternary structure of the complex .

How can the effects of Tim22 mutations on mitochondrial function be assessed?

Evaluating the impact of Tim22 mutations on mitochondrial function involves multiple approaches:

What factors might contribute to variability in Tim22 function across different experimental systems?

Several factors can contribute to experimental variability when studying Tim22 function:

• Oxidation State: The redox state of Tim22's conserved cysteine residues significantly affects its stability and function. Variations in the oxidative environment during preparation or experimentation can lead to inconsistent results .

• Expression Levels: Both under and overexpression of Tim22 can affect experimental outcomes. Oxidized Tim22 appears to have stronger interactions with Tim18 than reduced Tim22, and excess Tim22 that overflows from the TIM22 complex may not form a disulfide bond .

• Species Differences: While Tim22 function is conserved, there are structural and functional differences between yeast and mammalian systems that must be considered when comparing experimental results across species .

• Complex Integrity: The stability of the TIM22 complex is crucial for its function. Experimental conditions that disrupt complex integrity, such as detergent choice during solubilization, can significantly affect functional assays .

• Substrate Load: The functional importance of Tim22's structural features, such as disulfide bonds, becomes more pronounced when the TIM22 complex handles excess substrate proteins . Experimental variation in substrate levels can therefore lead to apparently conflicting results.

Understanding these variables can help researchers design more consistent and interpretable experiments when studying Tim22 function and the TIM22 import pathway.