Recombinant Mouse Mitochondrial inner membrane protein (Immt)
Description
Protein Structure and Sequence
Recombinant Mouse Mitochondrial inner membrane protein (Immt) is typically produced as a full-length protein consisting of 757 amino acids corresponding to the native mouse Immt protein (Q8CAQ8) . The complete amino acid sequence, as documented in product data, begins with MLRACQLSGVTVAAQSCLCGKFVLRPLRPCRRYSTSSSSGLTAGKIAGAGLLFVGGGIGG and continues through to QDWLKEARMTLETKQIVEILTAYASAVGIGTTQVQQE at the C-terminal end . This sequence information is critical for researchers seeking to understand the protein's functional domains and interaction sites with other mitochondrial components. The recombinant protein maintains the essential structural features of native Immt, making it suitable for in vitro studies of mitochondrial membrane architecture and function.
Recombinant Modifications and Tags
For research purposes, recombinant mouse Immt is typically produced with an N-terminal histidine tag (His-tag), which facilitates easier purification and detection in experimental settings . This modification allows for efficient isolation of the protein through affinity chromatography while preserving the functional characteristics of the native protein. The His-tagged recombinant Immt retains its ability to interact with mitochondrial membrane structures and partner proteins, making it valuable for biochemical and structural studies. When expressed in E. coli, this recombinant protein maintains high purity levels (typically >90% as determined by SDS-PAGE), ensuring reliability in experimental applications .
Expression Systems and Purification
Recombinant mouse Immt is commonly expressed in E. coli expression systems, which provide an efficient platform for producing large quantities of the protein . Alternative expression systems, such as silkworm-based platforms, have also been utilized for the production of mitochondrial inner membrane proteins, as they can efficiently express membrane proteins that may be challenging to produce in bacterial systems . The purification process typically involves affinity column chromatography targeting the N-terminal His-tag, followed by additional purification steps to achieve high purity. Successful solubilization of Immt proteins often requires detergents and high salt concentrations (>300 mM NaCl), reflecting the membrane-associated nature of the protein .
Physical and Chemical Properties
As a lyophilized powder, recombinant mouse Immt requires proper reconstitution before use in experimental applications . Recommended reconstitution involves dissolving the protein in deionized sterile water to concentrations of 0.1-1.0 mg/mL . For long-term storage, the addition of glycerol (5-50% final concentration) is advised to prevent protein degradation and maintain structural integrity . The stability of recombinant Immt is temperature-sensitive, and repeated freeze-thaw cycles should be avoided to preserve protein functionality. Working aliquots are best stored at 4°C for up to one week, while longer-term storage requires temperatures of -20°C or -80°C .
Role in Mitochondrial Structure
The native Immt protein (Mitofilin/Mic60) serves as a core component of the MICOS complex, which is critical for maintaining proper mitochondrial cristae structure . Located specifically at cristae junctions, Immt plays a fundamental role in organizing the inner mitochondrial membrane architecture. In mammalian systems, Mitofilin exists primarily in two isoforms of approximately 88 and 90 kDa, with four isoforms identified in brain tissue that differ in their isoelectric points due to post-translational modifications . The protein's structure includes an N-terminal anchor in the inner membrane, while the majority of the protein extends into the intermembrane space, enabling interactions with multiple protein partners .
Protein Interactions and Complexes
Immt/Mitofilin engages in numerous protein-protein interactions that are essential for mitochondrial structure and function. It directly interacts with Mic25, Mic19, and SAM50, forming the Mic60-Mic19-Mic25 subcomplex that establishes physical contacts between the inner and outer mitochondrial membranes . Additionally, Immt interacts with the translocase complexes of both the outer (TOM) and inner (TIM) mitochondrial membranes, influencing the import of precursor proteins into the mitochondria . Recent research has also identified an interaction between Mitofilin and Cyclophilin D, which plays a role in regulating the mitochondrial permeability transition pore (mPTP), a critical determinant in ischemia/reperfusion injury .
Pathophysiological Applications
The role of Immt in disease states, particularly in cardiac pathology, represents an important area of research utilizing recombinant Immt proteins. Studies with heterozygous Mitofilin knockout mice (Mitofilin+/-) have revealed that reduced Mitofilin expression leads to increased susceptibility to myocardial injury following ischemia/reperfusion . These mice exhibit decreased mitochondrial calcium retention capacity, increased reactive oxygen species (ROS) production, and dysregulated solute carrier function after ischemia/reperfusion injury . Such findings highlight potential therapeutic targets involving Immt for addressing cardiac ischemic damage and other mitochondrial-related pathologies.
Knockout Studies and Developmental Biology
Research utilizing Mitofilin knockout models has provided crucial insights into the protein's essential nature. Complete deletion of Mitofilin (homozygous knockout) is lethal in mouse offspring, underscoring the critical role of this protein in development and cellular viability . Interestingly, heterozygous mice with a single allele expression of Mitofilin display relatively normal phenotypes under non-stress conditions, suggesting that approximately half the normal protein level is sufficient for basic mitochondrial functions . These findings emphasize the importance of dosage-dependent effects in mitochondrial protein function and highlight the value of recombinant Immt for rescue experiments and complementation studies.
Reconstitution and Experimental Usage
When preparing recombinant mouse Immt for experimental applications, brief centrifugation of the vial is recommended prior to opening to ensure all material is collected at the bottom . Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL provides a working solution suitable for most applications . For experiments requiring higher protein stability, the addition of glycerol (typically 5-50% final concentration) is advised, with 50% being the standard recommendation for long-term storage . When using the reconstituted protein in membrane-based experiments, consideration must be given to the protein's natural affinity for lipid environments, particularly those containing cardiolipin, which is abundant in mitochondrial membranes.
Therapeutic Potential and Disease Models
The critical role of Immt in mitochondrial function makes it a potential target for therapeutic interventions in mitochondrial diseases. Research indicates that Mitofilin knockdown leads to mitochondrial cristae damage, increased reactive oxygen species production, and mitochondrial DNA release into the cytosol, activating inflammatory pathways that exacerbate ischemia/reperfusion injury . These findings suggest that stabilizing or enhancing Immt function could offer protective effects in conditions characterized by mitochondrial dysfunction. Recombinant mouse Immt provides a valuable tool for screening potential drug candidates that might modulate its activity or interactions with partner proteins.
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate your preference in the order notes. We will accommodate your request to the best of our ability.
Note: All proteins are shipped with standard blue ice packs. If dry ice shipping is required, please inform us in advance, as additional fees may apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Note: The complete sequence including tag sequence, target protein sequence and linker sequence could be provided upon request.
Target Background
- Mitofilin homeostasis, regulated by Yme1L, is central to the assembly of the MICOS complex. PMID: 26250910
- Mitofilin acts as a critical organizer of mitochondrial cristae morphology, making it indispensable for normal mitochondrial function. PMID: 15647377
- Research suggests that Uchl3 and mitofilin exhibit differential expression in the hippocampi of inbred senescence-accelerated mice compared to normally-aging mice. PMID: 18307031
Q&A
What is the molecular structure of IMMT and how does it contribute to mitochondrial membrane organization?
IMMT/Mitofilin is a core subunit of the MICOS (Mitochondrial Contact Site and Cristae Organizing System) complex, directly located adjacent to cristae junctions (CJ). The protein contains several key structural domains that contribute to its function. In humans, IMMT is anchored to the inner mitochondrial membrane (IMM) via its N-terminus, while most of the protein extends into the inner mitochondrial space (IMS) .
The protein's architecture includes:
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N-terminal membrane anchor domain
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Central coiled-coil domain (essential for protein-protein interactions)
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C-terminal mitofilin domain (crucial for building the MICOS complex)
This structural organization enables IMMT to form and maintain cristae junctions, which are critical for proper mitochondrial function and cristae morphology. Mouse IMMT shares significant homology with human IMMT, though species-specific structural variations should be considered when designing experiments.
How does IMMT interact with other components of the mitochondrial membrane system?
IMMT functions as a scaffold protein within the MICOS complex, facilitating interactions between multiple proteins at the cristae junctions. The protein's central coiled-coil domain specifically mediates these protein-protein interactions .
Research has shown that IMMT directly interacts with Disrupted-in-schizophrenia 1 (DISC1), which localizes to the inside of mitochondria. This interaction is functionally significant, as DISC1 is critical for the stability of IMMT/Mitofilin . When DISC1 function is reduced, mitochondrial dysfunction occurs, including:
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Decreased mitochondrial NADH dehydrogenase activities
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Reduced cellular ATP content
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Perturbed mitochondrial Ca²⁺ dynamics
These findings demonstrate that IMMT participates in a complex network of protein interactions that collectively maintain mitochondrial integrity and function.
What are the optimal conditions for expressing and purifying recombinant mouse IMMT?
Based on established protocols for recombinant human IMMT production, researchers working with mouse IMMT should consider the following parameters:
The purification protocol should include affinity chromatography using the His-tag, followed by size exclusion chromatography to achieve >95% purity as determined by SDS-PAGE . For functional studies, it's crucial to verify that the recombinant protein maintains its native conformation and activity.
How can researchers effectively measure mitochondrial function in relation to IMMT activity?
Several complementary approaches can be used to assess mitochondrial function when studying IMMT:
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Mitochondrial membrane potential assessment:
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Tetramethylrhodamine (TMRM) staining followed by flow cytometry analysis provides quantitative measurement of mitochondrial membrane potential
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Higher TMRM signals correlate with increased granzyme B and IFN-γ production in T cells, indicating a relationship between membrane potential and cellular function
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Mitochondrial mass measurement:
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Oxygen consumption rate (OCR) analysis:
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Complex I activity assay:
These methodologies can be combined to comprehensively evaluate how alterations in IMMT expression or function impact mitochondrial performance in various experimental contexts.
What approaches can be used to study IMMT's role in cristae remodeling?
Cristae remodeling is a critical process in mitochondrial adaptation to cellular demands, and IMMT plays a central role in this process. Researchers can investigate this relationship using:
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Electron microscopy techniques:
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Transmission electron microscopy (TEM) provides high-resolution imaging of cristae morphology
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Quantitative parameters to assess include cristae width, density, and junction diameter
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Immunogold labeling can localize IMMT at cristae junctions
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Genetic manipulation strategies:
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Functional correlations:
Research on NDUFS4 (a component of mitochondrial complex I) provides a methodological framework, as genetic overexpression of this protein in diabetic mice showed significant improvements in cristae morphology and mitochondrial dynamics . Similar approaches could be applied when studying IMMT's role in cristae organization.
How does IMMT dysfunction contribute to mitochondrial-related pathologies?
IMMT dysfunction has been implicated in various pathological conditions through several mechanisms:
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Disruption of cristae morphology:
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Abnormal cristae structure impairs respiratory chain complex organization and efficiency
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This can lead to reduced ATP production and increased reactive oxygen species generation
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Altered protein interactions:
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The interaction between IMMT and DISC1 is crucial for normal mitochondrial function
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Disruption of this interaction leads to decreased mitochondrial NADH dehydrogenase activities, reduced cellular ATP content, and perturbed mitochondrial calcium dynamics
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These changes may contribute to neurological disorders, as DISC1 is a schizophrenia-susceptibility gene
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Impact on metabolic adaptations:
Understanding these pathological mechanisms can guide therapeutic strategies targeting mitochondrial function through IMMT modulation.
What experimental models are most appropriate for investigating IMMT dysfunction in various disease contexts?
When selecting models, researchers should consider tissue-specific expression patterns of IMMT and its interacting partners, as well as the particular aspects of mitochondrial function relevant to the disease being studied.
How can innovative techniques like optogenetics be applied to study IMMT's role in mitochondrial dynamics?
Optogenetic approaches offer unprecedented temporal and spatial control for studying mitochondrial dynamics. Recent advances in "OptoMito-On" technology provide insights into how similar approaches might be applied to IMMT research:
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Spatiotemporal control of IMMT function:
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Light-responsive domains could be fused to IMMT to control its activity or interactions with binding partners
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This would allow precise manipulation of cristae organization in specific subcellular regions
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Real-time visualization:
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Combining optogenetic control with live-cell imaging techniques could reveal dynamic changes in cristae morphology
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Fluorescent protein tags on IMMT and interacting partners would enable monitoring of protein complex formation and disassembly
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Correlation with functional outcomes:
The development of "OptoMito-On" demonstrates the feasibility of remotely controlling mitochondrial metabolism with outstanding specificity and temporospatial resolution . Adapting these techniques to IMMT research could provide new insights into its dynamic roles.
What are the methodological considerations for measuring the effects of IMMT manipulation on mitochondrial respiratory complexes?
When investigating how IMMT affects mitochondrial respiratory complexes, researchers should consider:
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Comprehensive respiratory chain assessment:
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Proteomic approaches:
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Functional consequences:
These methodological considerations ensure a comprehensive understanding of how IMMT impacts not just mitochondrial structure but also its fundamental bioenergetic functions.
What are the most significant technical challenges in IMMT research, and how might they be addressed?
| Challenge | Potential Solutions | Implementation Considerations |
|---|---|---|
| Full-length protein expression | Use of specialized expression systems (insect cells, cell-free systems) | May require optimization of codon usage and removal of hydrophobic regions |
| Maintaining native conformation | Incorporation of stabilizing agents and appropriate detergents | Functional validation required to confirm proper protein folding |
| Distinguishing direct vs. indirect effects | Careful experimental design with appropriate controls | Use of specific binding-deficient mutants can isolate direct effects |
| In vivo assessment of IMMT function | Development of conditional and tissue-specific mouse models | Consider temporal control using inducible systems |
| Translation to human disease relevance | Comparative studies between mouse and human IMMT | Focus on conserved domains and functions |
What emerging research directions might advance our understanding of IMMT biology?
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Single-cell approaches:
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Single-cell proteomics and transcriptomics could reveal cell-specific roles of IMMT
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May identify previously unknown cell populations particularly dependent on IMMT function
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Integration with metabolomics:
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Comprehensive metabolomic profiling alongside IMMT manipulation
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Would connect structural changes to specific metabolic pathways affected
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Therapeutic targeting:
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Development of small molecules that stabilize IMMT or enhance its interactions with key partners
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Could represent novel approaches for treating diseases with mitochondrial dysfunction
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Cross-species comparative studies:
These emerging directions highlight the dynamic nature of IMMT research and its potential implications for understanding fundamental mitochondrial biology and disease pathogenesis.
