Recombinant Mitochondrial import inner membrane translocase subunit tim-22 (tim-22)

What is the TIM22 complex and what is its primary function in mitochondria?

The TIM22 complex is a specialized translocase responsible for the import of multiple hydrophobic carrier proteins that are subsequently folded into the inner mitochondrial membrane. This complex is essential for maintaining mitochondrial proteostasis, as these carrier proteins perform vital functions in cellular metabolism and homeostasis. The TIM22 complex serves as a protein insertase rather than a closed channel, with a structure that forms a lateral hydrophobic cave exposed to the lipid bilayer . This specialized architecture enables the complex to facilitate the lateral insertion of multi-pass transmembrane proteins into the inner mitochondrial membrane, which is critical for maintaining mitochondrial function and cellular viability.

What is the composition of the human TIM22 complex?

In mammalian cells, the TIM22 complex consists of at least six components:

• Tim22 - The core subunit with four transmembrane helices forming a partial pore

• Tim29 - A single transmembrane protein that stabilizes Tim22

• AGK (Acylglycerol Kinase) - A component specific to the mammalian complex

• Three Tim chaperones:

  • Tim9

  • Tim10a

  • Tim10b

These components assemble into a complex with specific stoichiometry, where the Tim9/Tim10a hexamer has a 3:3 molar ratio, while the Tim9/Tim10a/Tim10b hexamer has a 2:3:1 molar ratio . This precise arrangement is crucial for the complex's functionality in recognizing and inserting specific substrate proteins.

How are TIM22 complex substrates recognized and imported?

The TIM22 complex recognizes and imports specific substrates through a multi-step process:

• Hydrophobic carrier protein precursors are recognized and chaperoned by hexameric Tim complexes in the intermembrane space

• The Tim9/Tim10a/Tim10b hexamer serves as a dock, facing the intramembrane region of Tim22 to load precursors to the partial pore of Tim22

• The four transmembrane segments of Tim22 form a lateral gate that allows precursors to be inserted into the lipid bilayer

• Insertion is driven by membrane potential and facilitated by the unique architecture of the TIM22 complex

This process is highly selective, ensuring that only appropriate substrates are imported through this pathway, while other mitochondrial proteins are directed to different import pathways.

What is the detailed structural organization of the TIM22 complex?

Cryo-EM structural analysis at 3.7Å resolution reveals that the human TIM22 complex is approximately 100Å in height and 160Å in the longest dimension. Key structural features include:

• The core subunit Tim22 contains two helices (α1 and α2) connected by an extended loop, and four transmembrane segments (TM1-4)

• The Tim22 N-terminal helices protrude toward the intermembrane space and interact with the Tim9/Tim10a/Tim10b hexamer

• A disulfide bond between Cys69 and Cys141 stabilizes the conformations of TM1 and TM2 in Tim22

• Tim29 exhibits an extended conformation with a long N-terminal helix in the matrix, a single TM, an intermembrane space domain, and a C-terminal chaperone recruiting motif

• The Tim9/Tim10a/Tim10b hexamer is positioned at approximately 45° tilt relative to the membrane, not perpendicular

This intricate arrangement allows for precise coordination of substrate recognition, transfer, and insertion into the inner membrane.

How do the TIM chaperone hexamers contribute to TIM22 complex function?

The TIM chaperone hexamers play crucial roles in the TIM22 complex:

• The Tim9/Tim10a hexamer (3:3 ratio) primarily functions to bind and shield hydrophobic segments of precursor proteins during transit through the aqueous intermembrane space

• The Tim9/Tim10a/Tim10b hexamer (2:3:1 ratio) serves as a hub at the center of the TIM22 complex

• The Tim9/Tim10a/Tim10b hexamer is encircled by the N-terminus of Tim22, the middle portion of Tim29, AGK, and the Tim9/Tim10a chaperone

• Specific interactions stabilize the complex:

  • Helix α1 of Tim22 nestles in a greasy pocket formed by Tim9 and Tim10a

  • Hydrophobic residues (Leu28, Leu29, Leu32, Val33) from Tim22 interact with residues from Tim9 and Tim10a

  • Salt bridges and hydrogen bonds further reinforce these interactions

These interactions not only maintain the structural integrity of the complex but also create a pathway for guiding hydrophobic precursors from the intermembrane space into the inner membrane.

How does the TIM22 complex interact with mitochondrial quality control systems?

The TIM22 complex exhibits important functional relationships with mitochondrial quality control systems, particularly proteases like Yme1:

• Yme1 is an inner membrane metalloprotease that regulates protein quality control with both chaperone-like and proteolytic activities

• Genetic analyses indicate that impairment in the TIM22 complex can rescue respiratory growth defects of cells lacking Yme1

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

• Excessive levels of TIM22 pathway substrates appear to contribute to respiratory growth defects in cells lacking Yme1

• Compromising the TIM22 complex can compensate for the imbalance in mitochondrial proteostasis caused by the loss of Yme1

This functional crosstalk highlights the integrated nature of mitochondrial protein import and quality control systems, demonstrating how these pathways work together to maintain mitochondrial homeostasis.

What happens when TIM22 pathway substrate import is dysregulated?

Dysregulation of TIM22 pathway substrate import can have significant consequences:

• Excess of TIM22 pathway substrates leads to proteostatic stress and can ultimately result in cell death

• In cells lacking the quality control protease Yme1, accumulation of TIM22 substrates appears to contribute to respiratory growth defects

• Imbalances in TIM22 pathway substrate levels affect mitochondrial structural and functional integrity

• Mitochondrial protein import clogging can occur when substrate levels exceed the capacity of the import machinery, which has been identified as a potential mechanism of disease

These findings emphasize the importance of maintaining proper balance in mitochondrial protein import pathways and the critical role that the TIM22 complex plays in cellular homeostasis.

What methodologies are used to study the TIM22 complex structure?

Researchers employ several advanced methodologies to investigate TIM22 complex structure:

• Cryo-electron microscopy (Cryo-EM):

  • Sample preparation using vitrification on holey carbon grids

  • Imaging on transmission electron microscopes (e.g., FEI Titan Krios at 300 kV)

  • Data collection using direct electron detectors (e.g., Gatan K2 Summit)

  • Motion correction using MotionCor2 and defocus estimation using Gctf

• Structural model building and refinement:

  • De novo building of protein structures using COOT

  • Refinement against cryo-EM maps using PHENIX in real space with secondary structure restraints

  • Validation through Molprobity scores and Ramachandran plot statistics

• Complementary approaches:

  • X-ray crystallography for individual components (e.g., Tim9/Tim10a hexameric chaperone)

  • Crosslinking mass spectrometry to identify protein-protein interactions

  • Molecular dynamics simulations to understand conformational dynamics

These methodologies allow researchers to obtain detailed structural information about the TIM22 complex, which is crucial for understanding its mechanism of action.

What genetic and biochemical approaches are used to study TIM22 function?

Researchers employ various genetic and biochemical techniques to investigate TIM22 function:

• Genetic approaches:

  • Gene deletion/knockout studies (e.g., YME1 deletion in yeast)

  • Site-directed mutagenesis to create specific mutations (e.g., Val33Leu disease-related mutation in Tim22)

  • Genetic suppressor screens to identify functional relationships between genes

• Biochemical approaches:

  • Recombinant protein expression and purification

  • In vitro reconstitution of the TIM22 complex

  • Import assays using radiolabeled precursor proteins

  • Blue native polyacrylamide gel electrophoresis (BN-PAGE) to assess complex integrity

  • Protein stability assays to evaluate the effects of mutations or interacting partners

• Cell biology approaches:

  • Mitochondrial morphology assessment

  • Respiratory function measurements

  • Cellular growth assays under different conditions (e.g., fermentable vs. non-fermentable carbon sources)

These approaches have revealed important insights, such as the finding that Yme1 is essential for TIM22 complex stability and that impairment in the TIM22 complex can rescue respiratory growth defects in Yme1-deficient cells .

What disease-associated mutations have been identified in TIM22 components?

Several disease-associated mutations have been identified in TIM22 components:

• The Val33Leu mutation in Tim22 has been identified as disease-related . This mutation is located in helix α1, which interacts with the Tim9/Tim10a chaperone. The mutation likely affects this interaction, potentially disrupting the structure and function of the entire complex.

• Research suggests that TIM22 pathway dysfunction may contribute to various mitochondrial diseases through:

  • Impaired import of essential carrier proteins

  • Proteostatic stress due to accumulation of import substrates

  • Mitochondrial protein import clogging as a mechanism of disease

Understanding these disease mechanisms provides insights into potential therapeutic targets for mitochondrial disorders.

How can researchers modulate TIM22 complex activity for experimental purposes?

Researchers can modulate TIM22 complex activity through several approaches:

• Genetic modulation:

  • Knockdown/knockout of specific TIM22 complex components

  • Expression of dominant-negative mutants

  • Generation of hypomorphic alleles for partial loss of function

• Biochemical modulation:

  • Targeting the disulfide bond between Cys69 and Cys141 in Tim22

  • Modifying membrane potential to affect import efficiency

  • Altering the expression levels of TIM22 pathway substrates

• Combined approaches:

  • Simultaneous modulation of the TIM22 complex and interacting partners (e.g., YME1)

  • The finding that impairment in the TIM22 complex rescues respiratory growth defects of cells without Yme1 provides a strategy for genetic suppression studies

These approaches enable researchers to investigate the roles of the TIM22 complex in normal physiology and disease states.

What are the current technical challenges in studying TIM22-mediated protein import?

Researchers face several technical challenges when studying TIM22-mediated protein import:

• Reconstitution challenges:

  • Difficulty in recombinant expression and purification of full TIM22 complex

  • Maintaining the native conformation of hydrophobic membrane proteins

  • Recreating appropriate membrane environment for functional studies

• Structural challenges:

  • Capturing different conformational states during the import process

  • Determining the structure of the complex with bound substrate

  • Understanding the dynamic interactions between components during substrate translocation

• Functional challenges:

  • Distinguishing direct from indirect effects in complex mitochondrial networks

  • Isolating the specific contribution of individual components

  • Measuring real-time protein import kinetics in live cells

Addressing these challenges requires continued development of innovative experimental approaches and technologies.

What are the most promising future research directions for TIM22 studies?

Several promising research directions are emerging in the field of TIM22 studies:

• Structure-function relationships:

  • Determining high-resolution structures of the TIM22 complex with bound substrates

  • Investigating conformational changes during the import process

  • Understanding the molecular basis of substrate specificity

• Disease mechanisms:

  • Exploring the role of TIM22 dysfunction in various mitochondrial diseases

  • Investigating how mutations in TIM22 components affect protein import

  • Developing therapeutic strategies to address import defects

• Systems biology approaches:

  • Mapping the network of interactions between the TIM22 complex and other mitochondrial systems

  • Understanding how TIM22-mediated import is regulated in response to cellular stress

  • Exploring the evolutionary diversity of the TIM22 complex across different organisms

These research directions hold promise for advancing our understanding of mitochondrial protein import and its implications for health and disease.