Recombinant Debaryomyces hansenii Mitochondrial import inner membrane translocase subunit TIM14 (PAM18)

What is TIM14/PAM18 and what role does it play in mitochondrial protein import?

TIM14/PAM18 is an essential component of the mitochondrial import motor of the TIM23 translocase. It functions as a J-domain protein that stimulates the ATPase activity of mitochondrial Hsp70 (mtHsp70), enabling efficient protein translocation into the mitochondrial matrix. TIM14 is anchored in the inner mitochondrial membrane with its J-domain exposed to the matrix space . The protein is critical for the import of preproteins that require the action of mtHsp70, particularly those with an N-terminal matrix-targeting signal . Deletion or depletion of Tim14 results in severe defects in mitochondrial protein import, demonstrating its essential role in this process .

The PAM complex, which includes TIM14/PAM18, powers the release of imported proteins into the matrix through ATP hydrolysis via Hsp70. This complex consists of several proteins including Tim44, Tim15, Pam16, Pam18, Mge1, and Hsp70 . Within this machinery, TIM14/PAM18 activates mtHsp70, enabling it to function rapidly and in a regulated manner in the Tim44-mediated trapping of unfolded preproteins entering the matrix .

How does TIM14/PAM18 interact with other components of the mitochondrial import machinery?

TIM14/PAM18 forms specific interactions with multiple components of the TIM23 translocase complex:

• Interaction with Tim44 and mtHsp70: TIM14 interacts with Tim44 and mtHsp70 in an ATP-dependent manner. These interactions are crucial for the functioning of the import motor .

• Complex formation with Tim16: TIM14 forms a heterodimer with Tim16 (also known as Pam16), a J-like protein lacking the HPD motif. This interaction stimulates the activity of TIM14 .

• Association with the TIM23 complex: Co-immunoprecipitation experiments have revealed that TIM14 is physically associated with the TIM23 complex. Antibodies against Tim17 and Tim23 efficiently co-precipitate TIM14, along with Tim50 and Tim44 .

• Recruitment mechanism: Evidence suggests that Tim14 is recruited to the Tim17·Tim23 subcomplex by Tim44. In mitochondria depleted of Tim44, only minor amounts of Tim14 were co-precipitated with antibodies against Tim17 and Tim23 .

What is the functional significance of the TIM14-TIM16 complex?

The TIM14-TIM16 complex represents a critical regulatory unit within the mitochondrial import motor. Crystal structure analysis reveals that the conserved domains of these two proteins have virtually identical folds despite limited sequence similarity . This structural arrangement has significant functional implications:

• Regulatory mechanism: Tim16 (Pam16) lacks the HPD motif necessary for stimulating Hsp70 ATPase activity but forms a heterodimer with Pam18 and in doing so, stimulates the activity of Pam18 .

• Essential nature: Both Pam16 and Pam18 are essential for yeast viability, indicating their crucial role in mitochondrial function .

• Domain requirements: Truncation mutant studies indicate that the conserved matrix domain of Tim14, which includes the J-domain and approximately 10 additional residues at its N-terminal side, is sufficient to support growth of yeast cells in the absence of wild-type protein .

• N-terminal domain function: Interestingly, deletion of the N-terminal segment of Tim14, which extends into the intermembrane space in yeast, had virtually no effect on its function, despite suggestions that this domain might be essential for recruiting the import motor to the membrane-embedded part of the TIM23 translocase .

How does TIM14/PAM18 localization differ in plants compared to other eukaryotes?

Plants exhibit unique characteristics regarding the localization of TIM14/PAM18 homologs:

• Multiple orthologs: Plants contain multiple copies of Pam16 and Pam18 genes, suggesting functional specialization or redundancy .

• Dual targeting: While Pam16 orthologs (Pam16-1 and Pam16-2) are exclusively targeted to mitochondria, Pam18 orthologs (Pam18-1, Pam18-2, and Pam18-3) can be dual-targeted to both mitochondria and plastids, depending on the methodology used and tissue types investigated .

• Tissue-specific localization: The localization pattern of Pam18 orthologs varies based on experimental approaches and tissue types:

This dual targeting capability suggests a possible role for Pam18 co-chaperones in plastids, representing an evolutionary adaptation specific to plants.

What are the effects of TIM14 depletion on mitochondrial protein import?

Depletion of TIM14 has profound effects on mitochondrial protein import processes:

• General import defects: Mitochondria isolated from cells depleted of Tim14 showed strong defects in the import of most precursors using the TIM23 translocase pathway .

• Selective impact: Interestingly, the only precursors of the TIM23 translocase that were not significantly affected were those which do not need a functional mitochondrial import motor . This selective impact highlights the specific role of TIM14 in the motor function of the import machinery.

• Complex integrity: In Tim14-depleted mitochondria, antibodies against Tim17 and Tim23 were still able to pull down the Tim17·Tim23·Tim50 subcomplex together with Tim44, with a similar efficiency to wild-type mitochondria . This suggests that while TIM14 is a component of the TIM23 translocase, it is not required for the structural integrity of the core complex.

• Functional consequences: The depletion studies indicate that Tim14 is required for the efficient binding of mtHsp70 to incoming polypeptide chains and for release from Tim44 , highlighting its crucial role in the dynamic process of protein translocation.

What expression systems are suitable for producing recombinant TIM14/PAM18?

Based on the available information, E. coli serves as an effective expression system for recombinant TIM14/PAM18 production:

• E. coli expression: Recombinant full-length Debaryomyces hansenii TIM14/PAM18 protein has been successfully expressed in E. coli with an N-terminal His tag .

• Protein properties: The recombinant protein typically includes the full-length sequence (172 amino acids) and can be produced with high purity (>90% as determined by SDS-PAGE) .

• Storage and handling: The recombinant protein is often supplied as a lyophilized powder that should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, addition of 5-50% glycerol (final concentration) and aliquoting for storage at -20°C/-80°C is recommended. Repeated freeze-thaw cycles should be avoided .

• Buffer conditions: Tris/PBS-based buffer with 6% Trehalose at pH 8.0 has been successfully used as a storage buffer for the recombinant protein .

What experimental approaches can be used to study TIM14/PAM18 localization and function?

Several complementary approaches have been used to study the localization and function of TIM14/PAM18:

• In vitro protein import assays: This technique uses isolated organelles (mitochondria or plastids) to assess the import capability of proteins. For TIM14/PAM18, this approach has confirmed that Pam18 orthologs can be imported into both mitochondria and plastids .

• Transient biolistic transformation: This method involves introducing GFP-tagged constructs into cell suspensions and observing protein localization. This technique has been used to demonstrate the dual targeting of some Pam18 orthologs .

• Stable transgenic GFP-tagged lines: Creating stable transgenic plants expressing GFP-tagged proteins provides a more physiological context for studying protein localization. Both 35S promoter and native promoter constructs have been used to study TIM14/PAM18 targeting .

• Co-immunoprecipitation: This technique has been valuable for identifying interactions between TIM14 and other components of the TIM23 complex. Antibodies against Tim17 and Tim23 have successfully co-precipitated Tim14, Tim50, and Tim44 .

• Depletion studies: Selective depletion of TIM14 or other components of the import machinery in yeast has provided insights into their functional roles and interdependencies .

How can researchers assess the functional importance of specific domains in TIM14/PAM18?

Several experimental approaches have been employed to determine the functional significance of different TIM14/PAM18 domains:

• Truncation mutants: Creation of mutants lacking specific domains (e.g., the intermembrane space domain or the transmembrane domain) has helped identify the minimum functional unit of TIM14. Studies have shown that even a mutant containing only the conserved matrix domain was able to support growth of cells and functionally replace wild-type Tim14 .

• Site-directed mutagenesis: Mutations in specific motifs, such as the HPD motif in the J-domain, have demonstrated their essential role in TIM14 function. A mutation in the HPD motif of the J-domain is lethal, confirming its critical importance .

• Growth assays: Testing the ability of mutant versions of TIM14 to support growth of yeast cells in the absence of wild-type protein has been valuable for assessing functional importance. This approach has shown that deletion of the N-terminal segment of Tim14 had virtually no effect on its function in yeast .

• In vitro import assays with isolated mitochondria: This approach allows researchers to directly assess the impact of TIM14 modifications on protein import efficiency. Mitochondria isolated from cells depleted of Tim14 showed strong defects in the import of most precursors using the TIM23 translocase pathway .

What is known about the crystal structure of the TIM14-TIM16 complex?

The crystal structure of the TIM14-TIM16 complex provides important insights into its molecular architecture and function:

• Structural similarity: Despite limited sequence similarity, the conserved domains of Tim14 and Tim16 have virtually identical folds .

• Crystallization conditions: The complex has been crystallized in different space groups:

  • P2₁2₁2₁ for the Tim14-Tim16 complex

  • P4₃2₁2 for the Tim14-Tim16-K₂OsCl₆ complex

• Cell constants: For the P2₁2₁2₁ space group, cell constants of a=112 Å, b=115 Å were reported, though the c value is partially cut off in the available information .

• Functional implications: The structural analysis suggests that Tim16 (Pam16) regulates the activity of Tim14 (Pam18) through direct interaction, forming a heterodimeric regulatory unit within the import motor.

How do ATP-dependent interactions contribute to TIM14/PAM18 function?

ATP plays a crucial role in regulating the interactions and function of TIM14/PAM18 within the mitochondrial import motor:

• Tim14-Tim44-mtHsp70 interactions: Tim14 interacts with Tim44 and mtHsp70 in an ATP-dependent manner . These interactions are essential for the proper functioning of the import motor.

• Regulation of mtHsp70: Tim14 is required for the activation of mtHsp70, enabling this chaperone to act in a rapid and regulated manner in the Tim44-mediated trapping of unfolded preproteins entering the matrix .

• J-domain function: The J-domain of Tim14, with its essential HPD motif, is crucial for stimulating the ATPase activity of Hsp70 . This stimulation drives the ATP-dependent cycle of binding and release that powers protein translocation.

• ATP hydrolysis mechanism: The release of proteins into the matrix is powered by ATP hydrolysis via Hsp70 in the PAM complex, which consists of proteins Tim44, Tim15, Pam16, Pam18, Mge1, and Hsp70 .

How conserved is TIM14/PAM18 across different species?

TIM14/PAM18 shows significant conservation across eukaryotes, though with some notable variations:

• Evolutionary conservation: TIM14 genes are present in the genomes of virtually all eukaryotes, indicating their fundamental importance in mitochondrial function .

• Structural conservation: The J-domain and its crucial HPD motif are highly conserved, reflecting their essential functional role in protein import .

• Species-specific variations: While the core function is conserved, there are species-specific adaptations. For example:

  • Yeast Tim14 has an N-terminal domain extending into the intermembrane space that is dispensable for function

  • Plants have multiple Pam18 orthologs with varying localization patterns

• Dual targeting in plants: The ability of plant Pam18 orthologs to target both mitochondria and plastids represents an evolutionary adaptation specific to plants, possibly related to the presence of two endosymbiotic organelles .