Recombinant Yarrowia lipolytica Mitochondrial import inner membrane translocase subunit TIM21 (TIM21)

What is TIM21 and what is its fundamental role in mitochondrial function?

TIM21 is a component of the presequence translocase of the inner membrane (TIM23 complex) that plays a crucial role in the formation of translocation contact sites between the outer and inner mitochondrial membranes. The protein is anchored in the mitochondrial inner membrane by a single transmembrane domain with its C-terminal domain facing the intermembrane space. This spatial arrangement is critical for its function in facilitating protein import into mitochondria .

TIM21 serves as a molecular bridge between the translocase of the outer membrane (TOM complex) and the TIM23 complex, ensuring efficient transfer of preproteins between these two translocation machineries. This function is essential for the import of nuclear-encoded proteins with N-terminal presequences into mitochondria. The physical connection created by TIM21 ensures that proteins being translocated across the outer membrane can be directly received by the inner membrane machinery without being released into the intermembrane space .

Additionally, TIM21 has been shown to interact with components of the respiratory chain, particularly complexes III and IV, suggesting a dynamic role in coordinating protein import with respiratory chain biogenesis and function. This dual interaction capacity highlights TIM21's importance in maintaining mitochondrial homeostasis and function .

Why is Yarrowia lipolytica an effective model organism for studying mitochondrial proteins like TIM21?

Yarrowia lipolytica has emerged as a powerful model organism for mitochondrial research due to several distinct advantages. As an obligate aerobic yeast, Y. lipolytica has a respiratory chain structure that more closely resembles that of higher eukaryotes compared to fermentative yeasts like Saccharomyces cerevisiae. Its respiratory chain contains complexes I-IV, one "alternative" NADH-dehydrogenase (NDH2), and a non-heme alternative oxidase (AOX) .

A particularly useful feature of Y. lipolytica for mitochondrial studies is that complex I is essential in this organism. This is because the NADH binding site of its alternative NADH-dehydrogenase (NDH2) faces the mitochondrial intermembrane space rather than the matrix. This characteristic has allowed researchers to develop sophisticated genetic tools for studying complex I and related mitochondrial functions .

The genetic tractability of Y. lipolytica has been enhanced through the development of specialized tools, including deletion strains for several complex I subunits that can be complemented by shuttle plasmids carrying the deleted gene. The ability to attach affinity tags, such as a hexa-histidine tag, to specific proteins facilitates efficient purification of mitochondrial complexes. For example, tagging the NUGM (30 kDa) subunit allows fast and efficient purification of complex I by affinity chromatography .

How does TIM21 facilitate the formation of translocation contact sites at the molecular level?

The molecular mechanism by which TIM21 facilitates the formation of translocation contact sites involves specific interactions between its C-terminal domain and components of the TOM complex. Research has shown that the purified C-terminal domain of TIM21, which is exposed to the intermembrane space, does not bind to other components of the TIM23 complex but instead specifically interacts with the TOM complex at its trans site (the side facing the intermembrane space) .

This interaction creates a physical bridge between the TOM and TIM23 complexes, establishing translocation contact sites where the outer and inner membranes come into close proximity. These contact sites form dynamically regulated channels through which preproteins can be directly transferred from the TOM complex to the TIM23 complex without being released into the intermembrane space .

The formation of these contact sites is critical for the efficient import of preproteins with N-terminal presequences. When TIM21 associates with the TIM17:23 complex, it forms what has been termed the sorting and organization translocase (SORT) complex, which includes accessory proteins like Tim50. This configuration promotes the tethering of Tim21 to the outer membrane translocation pore and facilitates the insertion of proteins into the inner membrane. Importantly, TIM21 appears to have a direct role in the import and insertion of proteins into the inner membrane but not in the translocation of matrix-located proteins .

What experimental approaches can be used to study TIM21's interactions with respiratory chain complexes?

Several sophisticated experimental approaches can be employed to investigate TIM21's interactions with respiratory chain complexes:

Yeast Two-Hybrid (Y2H) Assays: This technique has been successfully used to detect protein-protein interactions between TIM21 and respiratory chain components. For example, research has shown interactions between AtTim21 (from Arabidopsis) and components of complex III, including the ubiquinol-cytochrome c reductase cytochrome c1 subunit (AtCyc1-1), MPPα, and the Rieske iron-sulfur protein (RISP). In these experiments, TIM21 is cloned into a bait vector and transformed into one yeast strain, while potential interaction partners are cloned into prey vectors and transformed into a mating-compatible strain. After mating, the diploid yeast cells are selected on appropriate media to identify positive interactions .

Affinity Purification Coupled with Mass Spectrometry: This approach involves tagging TIM21 with an affinity tag (such as a hexa-histidine tag) and using affinity chromatography to purify TIM21 along with its interaction partners. The purified protein complex can then be analyzed by mass spectrometry to identify the components. This method has been particularly useful in identifying novel interaction partners and confirming known associations .

Co-immunoprecipitation: This technique uses antibodies against TIM21 or its interaction partners to precipitate protein complexes from mitochondrial extracts. The precipitated proteins can then be analyzed by Western blotting or mass spectrometry to identify interaction partners.

Blue Native PAGE: This non-denaturing electrophoresis technique allows the separation of intact protein complexes and can be used to analyze the association of TIM21 with respiratory chain complexes. It can be combined with a second dimension SDS-PAGE to identify individual components of the complexes .

How can recombinant Y. lipolytica TIM21 be used for in vitro reconstitution experiments?

Recombinant Y. lipolytica TIM21 provides a valuable tool for in vitro reconstitution experiments aimed at understanding mitochondrial protein import mechanisms. The following methodological approach can be employed:

Protein Expression and Purification: The recombinant full-length Y. lipolytica TIM21 protein with an N-terminal His-tag can be expressed in E. coli and purified using affinity chromatography. The protein is typically obtained as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE .

Reconstitution Protocol: For functional reconstitution, the lyophilized protein should be centrifuged briefly before opening to bring the contents to the bottom. It can then be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% and aliquot for storage at -20°C/-80°C .

Liposome Reconstitution: For functional studies, the purified TIM21 protein can be incorporated into liposomes containing phospholipids, particularly phosphatidylcholine. Research has shown that adding 400-500 molecules of phosphatidylcholine per complex can significantly reactivate NADH:ubiquinone oxidoreductase activity in purified complexes that have lost most of their activity during purification .

Functional Assays: Once reconstituted, the protein can be used in various functional assays, including:

• Binding assays with purified TOM complex components to study translocation contact site formation

• Pull-down experiments to identify interaction partners

• In vitro protein import assays using isolated mitochondria or reconstituted systems to assess TIM21's role in protein translocation

What are the differences between TIM21 and TIM21-like proteins across different species?

Comparative analysis of TIM21 and TIM21-like proteins across different species reveals significant variations in structure, function, and essentiality:

Yeast vs. Plant TIM21: In Saccharomyces cerevisiae, Tim21 is non-essential, and its deletion does not result in a lethal phenotype. In contrast, in Arabidopsis thaliana, AtTim21 (encoded by At4g00026) is essential, and its deletion results in early seedling lethality. This difference suggests a more critical role for TIM21 in plant mitochondrial function compared to yeast .

TIM21-like Proteins in Plants: In addition to AtTim21, Arabidopsis contains two Tim21-like proteins (AtTim21-like 1 and AtTim21-like 2) that share structural similarities with Tim21 but may have distinct functions. Yeast two-hybrid assays have shown that these Tim21-like proteins interact with components of the TIM17:23 complex and respiratory chain subunits, suggesting roles similar to Tim21 .

The following table summarizes key interaction partners of AtTim21 and AtTim21-like proteins based on yeast two-hybrid assays:

This table illustrates the diverse interaction patterns of TIM21 and TIM21-like proteins, suggesting potential functional specialization among these proteins in plants .

What are the technical considerations for expressing and purifying recombinant Y. lipolytica TIM21?

Successful expression and purification of recombinant Y. lipolytica TIM21 requires attention to several technical considerations:

Expression System: E. coli has been successfully used as an expression host for recombinant Y. lipolytica TIM21. The protein can be expressed with an N-terminal His-tag to facilitate purification. The expression construct typically includes the mature protein sequence (amino acids 77-269) without the native mitochondrial targeting sequence, as this would be processed during import in the native context .

Purification Strategy: Affinity chromatography using the His-tag is the primary purification method. This can be followed by additional purification steps such as size exclusion chromatography or ion-exchange chromatography if higher purity is required. The purified protein typically has greater than 90% purity as determined by SDS-PAGE .

Buffer Conditions: The protein is typically stored in a Tris-based buffer at pH 8.0 with 6% trehalose. This buffer composition helps maintain protein stability during storage. For long-term storage, the protein is often provided as a lyophilized powder .

Reconstitution Protocol: When reconstituting the lyophilized protein, it is recommended to briefly centrifuge the vial to bring the contents to the bottom, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage of the reconstituted protein, adding glycerol to a final concentration of 5-50% and aliquoting for storage at -20°C/-80°C is recommended .

Stability Considerations: Repeated freezing and thawing should be avoided as it can lead to protein denaturation and loss of activity. Working aliquots can be stored at 4°C for up to one week. For extended storage, the protein should be kept at -20°C or -80°C .

How can mutational analysis of TIM21 provide insights into its function?

Mutational analysis of TIM21 is a powerful approach for understanding its structure-function relationships and has been successfully applied in research on mitochondrial import mechanisms. This methodology involves introducing specific mutations into the TIM21 gene and analyzing the effects on protein function, interactions, and physiological outcomes.

The established genetic tools for Y. lipolytica provide an excellent platform for site-directed mutagenesis of TIM21. Researchers can target specific amino acid residues based on sequence conservation, predicted functional domains, or structural features. For example, mutations can be introduced in the transmembrane domain to assess its role in membrane anchoring, or in the C-terminal domain to identify residues critical for interaction with the TOM complex .

Once mutant constructs are generated, they can be expressed in TIM21 deletion strains to assess their ability to complement the deletion phenotype. This approach has been successfully used with other mitochondrial components in Y. lipolytica, allowing for the identification of functionally important amino acids. The characterization of these mutations can provide valuable insights into the mechanism of TIM21 function in translocation contact site formation and protein import .

Additionally, mutational analysis can be combined with protein interaction studies, such as yeast two-hybrid assays or co-immunoprecipitation, to identify specific residues involved in interactions with the TOM complex or respiratory chain components. This approach can help delineate the molecular basis of these interactions and their functional significance .

What role does TIM21 play in connecting protein import with respiratory chain assembly?

TIM21 serves as a multifunctional adaptor that connects the protein import machinery with respiratory chain assembly, suggesting a coordinated regulation of these two essential mitochondrial processes. Evidence from both yeast and plant systems indicates that TIM21 physically interacts with components of both the TIM23 complex and respiratory chain complexes III and IV .

In yeast, Tim21 has been shown to physically associate with both components of the TIM17:23 complex and respiratory subunits of complex III and IV, including cytochrome c1, Rieske Fe/S protein, and cox4. Similarly, in Arabidopsis, AtTim21 interacts with the TIM17:23 complex and Complex III. These interactions suggest that TIM21 may play a role in coordinating the import of nuclear-encoded respiratory chain components with their assembly into functional complexes .

The functional significance of these interactions is highlighted by studies of AtTim21 overexpression in Arabidopsis, which resulted in increased cell numbers, cell size, and ATP production. Additionally, the transcript abundance of complex III, IV, and ATP synthase subunits was up-regulated in these overexpression lines. These findings suggest that TIM21 may not only facilitate the import of respiratory chain components but also influence their expression and assembly into functional complexes .

A proposed model for TIM21's dual role involves its dynamic association with different protein complexes. When associated with the TIM17:23 complex, TIM21 may facilitate the import of preproteins through translocation contact sites. Subsequently, it may associate with respiratory chain complexes to promote the assembly of newly imported components into functional complexes. This dynamic shuttling between different complexes would allow TIM21 to coordinate protein import with respiratory chain assembly, ensuring efficient mitochondrial biogenesis and function .

How can researchers design experiments to study the evolutionary conservation of TIM21 function?

Designing experiments to study the evolutionary conservation of TIM21 function requires a comparative approach that examines TIM21 structure, interactions, and function across different species. Here is a methodological framework for such studies:

Complementation Studies: Test functional conservation by expressing TIM21 from different species in a yeast TIM21 deletion strain to assess whether they can complement the deletion phenotype. For example, researchers could express Arabidopsis AtTim21 or human TIM21 in a Y. lipolytica TIM21 deletion strain and assess growth, respiratory function, and protein import efficiency .

Domain Swap Experiments: Create chimeric proteins by swapping domains between TIM21 proteins from different species to identify which regions are functionally conserved and which have species-specific functions. For instance, the transmembrane domain or the C-terminal domain of Y. lipolytica TIM21 could be replaced with the corresponding domains from Arabidopsis or human TIM21 .

Interaction Studies: Perform comparative interaction studies using techniques such as yeast two-hybrid assays, co-immunoprecipitation, or pull-down experiments with recombinant proteins to assess whether TIM21 from different species interacts with the same partner proteins. This would reveal conservation of interaction networks across evolution .

The following table summarizes key experimental approaches for studying TIM21 evolutionary conservation:

What are the best approaches for studying TIM21's role in mitochondrial disease models?

Studying TIM21's role in mitochondrial disease models requires a multifaceted approach that integrates molecular, cellular, and physiological techniques. Here are methodological recommendations for such investigations:

Gene Editing and Mutagenesis: CRISPR-Cas9 technology can be used to introduce disease-associated mutations in the TIM21 gene or to create knockout/knockdown models in cellular or animal systems. This approach allows for the direct assessment of how TIM21 dysfunction contributes to mitochondrial disease phenotypes .

Patient-Derived Cell Models: Fibroblasts or induced pluripotent stem cells (iPSCs) from patients with mitochondrial diseases can be analyzed for alterations in TIM21 expression, localization, or function. These cells can also be used to test the effects of restoring normal TIM21 function through gene therapy or other interventions .

Mitochondrial Functional Assays: Comprehensive assessment of mitochondrial function in disease models should include measurements of:

• Protein import efficiency using radiolabeled or fluorescently labeled precursor proteins

• Respiratory chain complex activity and assembly

• Mitochondrial membrane potential

• ATP production

• Reactive oxygen species (ROS) generation

• Mitochondrial morphology and dynamics

Interactome Analysis: Quantitative proteomics approaches, such as SILAC (Stable Isotope Labeling with Amino acids in Cell culture) combined with affinity purification and mass spectrometry, can be used to identify changes in TIM21's interaction partners in disease states compared to healthy controls. This can reveal alterations in protein import pathways or respiratory chain assembly that contribute to disease pathogenesis .

Rescue Experiments: Testing whether overexpression of wild-type TIM21 or TIM21 from different species can rescue disease phenotypes in cellular or animal models can provide insights into therapeutic strategies. Additionally, testing small molecules that modulate TIM21 function or its interactions could identify potential therapeutic approaches .

The integration of these methodological approaches can provide a comprehensive understanding of TIM21's role in mitochondrial disease and potentially identify novel therapeutic targets for intervention.

What are the emerging trends in TIM21 research and potential applications?

Research on TIM21 and related proteins is evolving rapidly, with several emerging trends and potential applications on the horizon. The increasing recognition of TIM21's dual role in protein import and respiratory chain assembly has opened new avenues for investigation into the coordinated regulation of these essential mitochondrial processes .

One emerging trend is the exploration of TIM21's potential role in mitochondrial stress responses and quality control. Given its interaction with both the protein import machinery and respiratory chain components, TIM21 is strategically positioned to sense and respond to changes in mitochondrial function. Future research may uncover regulatory mechanisms involving TIM21 that adjust protein import rates in response to changes in respiratory chain activity or mitochondrial stress .

Another promising direction is the investigation of TIM21 as a potential therapeutic target for mitochondrial diseases. The finding that overexpression of AtTim21 in Arabidopsis led to increased ATP production suggests that modulating TIM21 levels or activity could potentially enhance mitochondrial function in disease states. This could be particularly relevant for diseases characterized by defects in respiratory chain complex assembly or activity .

The development of high-resolution structural studies of TIM21 and its complexes represents another important frontier. While functional studies have provided valuable insights into TIM21's role, detailed structural information would significantly enhance our understanding of the molecular mechanisms underlying translocation contact site formation and TIM21's interactions with various partner proteins .