Recombinant Human Mitochondrial inner membrane organizing system protein 1 (MINOS1)

What is MINOS1 and what is its role in mitochondria?

MINOS1 (Mitochondrial Inner Membrane Organizing System protein 1) is a highly conserved protein found in the mitochondrial inner membrane that plays a critical role in maintaining proper mitochondrial morphology. It is an integral component of the MICOS complex (Mitochondrial Contact Site and Cristae Organizing System), previously known as the mitofilin/Fcj1 complex. This complex is responsible for the organization of the mitochondrial inner membrane, particularly the formation and maintenance of cristae junctions (CJs), which are critical structures connecting the inner boundary membrane to the cristae membranes .

MINOS1 is essential for proper cristae morphology, as evidenced by studies showing that its depletion or mutation leads to altered mitochondrial inner membrane organization. Mitochondria lacking MINOS1 exhibit disrupted cristae structure and compromised mitochondrial function. The protein contributes to the structural integrity of the MICOS complex, which serves as a physical scaffold for cristae junction formation .

What is the structural organization of human MINOS1?

Human MINOS1 (also known as C1orf151 in earlier literature) is a small membrane protein characterized by two predicted transmembrane segments separated by a short stretch of charged residues. Unlike many mitochondrial proteins, MINOS1 lacks an appreciable N-terminal presequence .

The protein adopts a specific topology in the inner mitochondrial membrane, with both its N- and C-termini exposed to the intermembrane space (IMS). This orientation has been confirmed through protease protection analyses, where the C-terminus becomes accessible to proteases only after disruption of the outer membrane through osmotic swelling .

MINOS1 contains a glycine-rich region reminiscent of Atp20 (a dimerization factor for F1FoATPase), though unlike Atp20, MINOS1 is not associated with the F1FoATPase complex. This glycine-rich motif may be involved in protein-protein interactions within the MICOS complex. The protein is highly conserved among eukaryotes, highlighting its fundamental importance in mitochondrial function .

How is MINOS1 localized within mitochondria?

The subcellular localization of MINOS1 has been determined through multiple complementary approaches. Immunofluorescence microscopy with anti-MINOS1 antibodies in Vero cells reveals a primarily mitochondrial distribution pattern that overlaps with known mitochondrial markers such as cyclophilin D, confirming its mitochondrial localization .

To determine the sub-mitochondrial localization, biochemical fractionation analyses have been performed in HEK293T cells. These experiments show that MINOS1 is resistant to protease treatment in intact mitochondria but becomes accessible to proteases when the outer membrane is disrupted by osmotic swelling to create mitoplasts. This behavior is similar to that of TIM23, an established inner membrane protein with domains exposed to the intermembrane space .

Further characterization by alkaline extraction demonstrates that MINOS1 remains in the membrane fraction under high pH conditions, while peripheral membrane proteins such as TACO1 are released into the supernatant. This resistance to alkaline extraction confirms that MINOS1 is an integral membrane protein rather than merely associated with the membrane . Collectively, these findings establish MINOS1 as an integral protein of the mitochondrial inner membrane with exposure to the intermembrane space.

What are the components of the MICOS complex and how does MINOS1 interact with them?

The MICOS complex is a large protein assembly (>1 MDa) that includes several core components. Based on the provided research, we know that the complex includes:

• MINOS1/Mio10 (the focus of our discussion)

• MINOS2/Mitofilin (known as Fcj1 in yeast)

• MINOS3/CHCHD3

• QIL1 (a recently identified component necessary for complex integrity)

• MIC10 (which requires QIL1 for proper integration into the complex)

• Additional proteins including HSPA9 and DnaJC11

MINOS1 interacts directly with other MICOS components, particularly MINOS2/Mitofilin. This interaction has been confirmed through co-immunoprecipitation experiments coupled with mass spectrometry analysis. When MINOS1-containing complexes were isolated from human HEK293T cells following metabolic labeling (SILAC approach), Mitofilin/MINOS2 was recovered as a significant interactor. Similarly, in yeast, Mio10 (the yeast ortholog of MINOS1) associates with Fcj1 (the yeast ortholog of MINOS2/Mitofilin) .

The interactions within the MICOS complex are critical for its function. Disruption of these interactions, such as through the loss of QIL1, leads to the complex falling apart, resulting in compromised cristae junction integrity and altered mitochondrial morphology . The MICOS complex is thought to function as a physical scaffold for cristae junction formation, and some evidence suggests it may also be involved in contact site formation between the inner and outer mitochondrial membranes .

How does MINOS1 contribute to mitochondrial cristae formation?

MINOS1 plays a crucial role in maintaining proper cristae morphology through its function within the MICOS complex. Research has demonstrated that loss of MINOS1/Mio10 leads to significant alterations in mitochondrial inner membrane organization .

In yeast, cells lacking Mio10 display substantially altered mitochondrial morphology, as observed through fluorescence live-cell imaging. At the ultrastructural level, mitochondria from Mio10-deficient cells show a dramatic loss of inner membrane organization. Instead of forming the typical cristae structures, the inner membrane may reorganize into concentric rings resembling an onion-like structure .

The mechanism by which MINOS1 contributes to cristae formation appears to be through its participation in the MICOS complex, which is essential for cristae junction formation. Cristae junctions are narrow neck-like structures that connect the cristae membranes to the inner boundary membrane. The MICOS complex acts as a structural scaffold that stabilizes these junctions and helps maintain the characteristic folded morphology of the inner membrane .

While MINOS1 contains a glycine-rich region reminiscent of Atp20 (a dimerization factor for F1FoATPase involved in cristae formation), it functions independently of the F1FoATPase complex. Despite extensive investigation, no physical association between MINOS1/Mio10 and the F1FoATPase has been detected in either human or yeast mitochondria. This suggests that MINOS1 contributes to cristae formation through a mechanism distinct from the F1FoATPase oligomerization pathway .

What phenotypes are observed when MINOS1 is depleted or mutated?

Depletion or mutation of MINOS1 results in several distinctive phenotypes that highlight its importance in mitochondrial structure and function:

• Altered Mitochondrial Morphology: Loss of MINOS1/Mio10 leads to significantly changed mitochondrial morphology as observed by fluorescence microscopy. The normally tubular or elongated mitochondrial network may appear fragmented or otherwise abnormal .

• Disrupted Cristae Structure: At the ultrastructural level (as observed by electron microscopy), mitochondria lacking MINOS1 show severe disorganization of the inner membrane. The typical cristae structure is lost, and the inner membrane may form concentric rings resembling an onion-like structure or other aberrant conformations .

• Growth Defects: In yeast, mio10-mutant cells exhibit impaired growth on non-fermentable carbon sources such as glycerol, which require functional mitochondrial respiration. This indicates compromised mitochondrial function and oxidative phosphorylation capacity .

• MICOS Complex Disassembly: Loss of MINOS1 leads to destabilization of the entire MICOS complex, as it is an integral component required for proper complex assembly and maintenance .

• Impact on Mitochondrial Function: The structural abnormalities resulting from MINOS1 depletion are associated with compromised mitochondrial function, affecting processes such as oxidative phosphorylation, which rely on proper inner membrane organization .

These phenotypes underscore the essential role of MINOS1 in maintaining mitochondrial inner membrane architecture and function. The observed structural defects explain the functional impairments, as proper cristae organization is necessary for efficient oxidative phosphorylation and other mitochondrial processes.

What methods are effective for studying MINOS1 localization and topology?

Several complementary approaches have proven effective for investigating MINOS1 localization and topology:

These methods, when used in combination, provide robust evidence for the localization and topology of MINOS1 as an integral inner membrane protein with both termini exposed to the intermembrane space.

What techniques are used to investigate MINOS1 protein interactions within the MICOS complex?

Several sophisticated techniques have been employed to investigate the protein interactions of MINOS1 within the MICOS complex:

• Co-immunoprecipitation (Co-IP): This foundational technique uses antibodies against MINOS1 to pull down the protein along with its interaction partners from detergent-solubilized mitochondria. Digitonin is the preferred detergent as it maintains native protein complexes in their oligomeric state. The precipitated proteins can then be identified by Western blotting using antibodies against suspected interaction partners .

• Quantitative Proteomics with SILAC: Stable Isotope Labeling with Amino acids in Cell culture (SILAC) coupled with immunoprecipitation provides a powerful approach for identifying specific protein interactions while minimizing false positives:

  • Cells are metabolically labeled with heavy or light isotopes of amino acids

  • Label-swap experiments (switching the labeling scheme between control and experimental conditions) further increase stringency

  • Following immunoprecipitation, samples are analyzed by high-resolution LC-MS/MS

  • Proteins specifically enriched in both forward and reverse experiments are considered true interactors
    This approach identified MINOS2/Mitofilin, MINOS3/CHCHD3, HSPA9, and DnaJC11 as interaction partners of MINOS1 .

• Blue Native PAGE (BN-PAGE): This technique separates native protein complexes according to their size while maintaining protein-protein interactions. It has been used to demonstrate that MINOS1 is part of a large protein complex (>1 MDa) .

• Affinity Purification with Tagged Proteins: In yeast studies, ZZ-tagged versions of known MICOS components have been used to pull down the complex and identify interaction partners. For example, a ZZ tag on Atp20 was used to investigate potential interactions with Mio10 .

• Mass Spectrometry Analysis: Following protein separation by SDS-PAGE, in-gel digestion with trypsin, and nanoflow high-performance liquid chromatography coupled to mass spectrometry (nanoLC-MS/MS) provides definitive identification of proteins in complexes. Data analysis tools such as MaxQuant, Mascot, and Scaffold are used to process the raw MS data and quantify protein abundances .

These complementary approaches have collectively established the protein interaction network of MINOS1 within the MICOS complex, revealing its associations with key components involved in cristae junction formation and inner membrane organization.

How can recombinant MINOS1 be expressed and purified for functional studies?

While the search results don't specifically address recombinant MINOS1 expression and purification, we can outline a methodological approach based on standard procedures for membrane proteins and information about MINOS1's properties:

• Expression System Selection:

  • Bacterial systems (E. coli): May be challenging due to MINOS1 being a eukaryotic membrane protein

  • Yeast expression systems (S. cerevisiae or P. pastoris): Offer advantages for mitochondrial proteins

  • Insect cell expression (Baculovirus): Provides eukaryotic processing capabilities

  • Mammalian cell expression: Ensures proper folding and post-translational modifications

• Construct Design:

  • Addition of affinity tags (His6, GST, or FLAG) for purification

  • Consideration of tag placement (N- or C-terminus) based on MINOS1's topology (both termini in the intermembrane space)

  • Inclusion of a protease cleavage site for tag removal

  • Codon optimization for the chosen expression system

• Solubilization Strategy:

  • Selection of appropriate detergents is critical for membrane proteins

  • Digitonin has been successfully used to maintain MINOS1 in native complexes and would be a good starting point

  • Other mild detergents like DDM (n-dodecyl β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol) might be alternatives

• Purification Protocol:

  • Initial capture using affinity chromatography based on the chosen tag

  • Size exclusion chromatography to separate monomeric protein from aggregates or complexes

  • Optional ion exchange chromatography for further purification

  • For structural studies, detergent exchange or reconstitution into nanodiscs or liposomes may be necessary

• Quality Control:

  • SDS-PAGE and Western blotting to verify protein identity and purity

  • Circular dichroism spectroscopy to assess secondary structure

  • Thermal stability assays to determine protein folding and stability

  • Functional assays to confirm biological activity

• Reconstitution for Functional Studies:

  • Incorporation into liposomes or nanodiscs to recreate a membrane environment

  • Co-expression or reconstitution with other MICOS components to study complex formation

  • Negative stain electron microscopy or cryo-EM to analyze complex structure

This methodological approach would need to be optimized specifically for MINOS1, taking into account its hydrophobicity, size, and interaction partners. The successful purification of functional recombinant MINOS1 would provide valuable material for structural studies, in vitro reconstitution of the MICOS complex, and mechanistic investigations of cristae formation.

How does MINOS1 coordination with other MICOS components regulate cristae remodeling during cellular stress?

Cristae remodeling is a dynamic process that occurs during various cellular conditions, including apoptosis, mitophagy, and metabolic stress. While the provided search results don't directly address MINOS1's role in stress-induced cristae remodeling, we can outline a methodological approach to investigate this question:

• Experimental Design for Stress Conditions:

  • Researchers could subject cells to various stressors (oxidative stress, metabolic stress, hypoxia)

  • Time-course experiments would capture the dynamics of cristae remodeling

  • Live-cell imaging with fluorescently tagged MINOS1 and other MICOS components could track their redistribution during stress

  • Fixed cell analysis with super-resolution microscopy at different time points would provide detailed structural information

• Investigation of Post-translational Modifications:

  • Phosphorylation, acetylation, or ubiquitination of MICOS components might regulate complex assembly/disassembly during stress

  • Mass spectrometry analyses after stress induction could identify modifications on MINOS1

  • Creation of phosphomimetic or phospho-deficient mutants would help determine the functional significance of identified modifications

• Interaction Dynamics Analysis:

  • Proximity labeling techniques (BioID or APEX) could identify stress-specific interaction partners

  • FRET (Förster Resonance Energy Transfer) or FLIM (Fluorescence Lifetime Imaging Microscopy) could measure changes in protein-protein interactions within the MICOS complex during stress

  • Quantitative co-immunoprecipitation at different stress time points would reveal how the composition of MICOS complexes changes

• Functional Studies:

  • Comparison of wild-type cells with MINOS1-depleted cells under stress conditions

  • Rescue experiments with wild-type vs. mutant MINOS1 to identify domains critical for stress response

  • Analysis of how MINOS1 depletion affects mitochondrial functions during stress (membrane potential, ATP production, ROS generation)

This methodological framework would help researchers understand how MINOS1 and the MICOS complex respond to cellular stress and contribute to the dynamic remodeling of cristae, which is essential for mitochondrial adaptation to changing cellular demands.

What is the relationship between MINOS1 dysfunction and mitochondrial disease pathology?

The relationship between MINOS1 dysfunction and mitochondrial disease represents an important area for investigation. While the search results don't directly address MINOS1-related diseases, we can outline methodological approaches to study this connection:

• Patient Sample Analysis:

  • Sequencing of MINOS1 in patients with unexplained mitochondrial disorders

  • Analysis of MINOS1 expression levels and MICOS complex integrity in patient fibroblasts or muscle biopsies

  • Ultrastructural examination of mitochondria from patient samples to detect cristae abnormalities

  • Functional assessment of mitochondria from patients (respiratory chain activity, membrane potential)

• Disease Modeling:

  • Generation of patient-specific iPSCs (induced pluripotent stem cells) harboring MINOS1 mutations

  • Differentiation into relevant cell types (neurons, muscle cells, cardiomyocytes)

  • CRISPR/Cas9-mediated introduction of disease-associated mutations in cell lines

  • Creation of animal models (mouse, zebrafish, Drosophila) with MINOS1 mutations or tissue-specific knockouts

• Mechanistic Studies:

  • Investigation of how MINOS1 mutations affect MICOS complex assembly and stability

  • Analysis of downstream effects on mitochondrial functions (respiratory chain complexes, mtDNA maintenance)

  • Examination of cellular stress responses (unfolded protein response, mitophagy, apoptosis)

  • Study of tissue-specific manifestations of MINOS1 dysfunction

• Therapeutic Exploration:

  • Testing whether overexpression of wild-type MINOS1 can rescue disease phenotypes

  • Investigation of compounds that might stabilize mutant MINOS1 or compensate for its dysfunction

  • Examination of whether targeting other MICOS components could compensate for MINOS1 deficiency

  • Exploration of broader mitochondrial therapies (antioxidants, metabolic modifiers) in the context of MINOS1 dysfunction

One important clue from the search results is the observation that in some mitochondrial diseases, "the inner membrane of a mitochondrion is no longer folded; instead, the membrane may form concentric rings like the layers of an onion" . This description matches the phenotype observed in MINOS1/Mio10-deficient mitochondria, suggesting that MINOS1 dysfunction could potentially contribute to such disease presentations. A systematic investigation using the approaches outlined above would help establish the precise role of MINOS1 in mitochondrial disease pathology.

How do evolutionary differences in MINOS1 across species reflect functional adaptations in mitochondrial architecture?

The search results indicate that MINOS1/Mio10 is highly conserved across eukaryotes , suggesting fundamental importance in mitochondrial function. A methodological approach to investigate evolutionary adaptations would include:

• Comparative Genomic Analysis:

  • Comprehensive phylogenetic analysis of MINOS1 sequences across diverse eukaryotic lineages

  • Identification of conserved domains, motifs, and residues that might be functionally critical

  • Detection of lineage-specific adaptations or accelerated evolution in specific taxa

  • Correlation of sequence variations with ecological, metabolic, or physiological traits

• Structure-Function Studies Across Species:

  • Expression of MINOS1 homologs from different species in a common cellular background

  • Cross-species complementation experiments to test functional conservation

  • Creation of chimeric proteins combining domains from different species to map functional regions

  • Structural analysis (if available) to compare folding and interaction surfaces across species

• Mitochondrial Morphology Comparison:

  • Ultrastructural analysis of mitochondria across species with focus on cristae architecture

  • Quantitative analysis of cristae density, size, and junction characteristics

  • Correlation of MINOS1 sequence features with cristae morphological parameters

  • Investigation of tissue-specific MINOS1 variants and corresponding mitochondrial morphologies

• Experimental Evolutionary Approaches:

  • Directed evolution experiments to identify adaptive mutations in MINOS1 under different selective pressures

  • Resurrection of ancestral MINOS1 sequences to test functional properties in modern cells

  • Testing how MINOS1 variants perform under conditions mimicking different evolutionary environments (temperature, oxygen levels, metabolic substrates)

This evolutionary perspective could provide valuable insights into both the fundamental mechanisms of cristae formation and the specialized adaptations that have occurred in different lineages throughout eukaryotic evolution.

What are the challenges in studying MINOS1-protein interactions and how can they be overcome?

Studying MINOS1 interactions presents several technical challenges, with methodological solutions that researchers can employ:

• Challenge: Membrane Protein Solubilization

  • MINOS1 is an integral membrane protein with two transmembrane domains, making solubilization while preserving native interactions difficult

  • Solution: Careful optimization of detergent conditions is critical. The search results indicate that digitonin has been successfully used to maintain MINOS1 in its native complexes . Testing a panel of mild detergents (LMNG, DDM, GDN) at various concentrations and solubilization times can help identify optimal conditions.

• Challenge: Distinguishing Direct from Indirect Interactions

  • MINOS1 is part of a large protein complex (>1 MDa), making it difficult to determine which interactions are direct versus indirect

  • Solution: Cross-linking mass spectrometry (XL-MS) with short-distance cross-linkers can capture direct protein-protein contacts. In vitro binding assays with purified components can confirm direct interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map interaction surfaces between MINOS1 and its partners.

• Challenge: Detecting Transient or Dynamic Interactions

  • Some interactions within the MICOS complex may be transient or condition-dependent

  • Solution: Proximity labeling approaches like BioID or APEX2 can capture even transient interactions in living cells. Time-resolved analyses after stimuli that induce cristae remodeling can reveal dynamic changes in the interaction network.

• Challenge: Functional Validation of Interactions

  • Determining the functional significance of identified interactions is often difficult

  • Solution: Structure-guided mutagenesis of specific interaction interfaces followed by functional assays. CRISPR-based approaches to introduce mutations that specifically disrupt individual interactions rather than eliminating the entire protein. In vitro reconstitution of minimal interacting components to test functional outcomes.

• Challenge: Complex Heterogeneity

  • The MICOS complex may exist in different subassemblies or states

  • Solution: Blue Native PAGE combined with second-dimension SDS-PAGE can resolve different subcomplexes. Single-particle cryo-EM approaches can potentially identify and characterize different conformational states. Gradient ultracentrifugation can separate complexes based on size for further analysis.

The methodological solutions outlined above can help researchers overcome the significant challenges in studying MINOS1 interactions, leading to a more comprehensive understanding of how this protein functions within the MICOS complex to maintain mitochondrial inner membrane architecture.

How can researchers effectively model and visualize MINOS1's role in cristae junction formation?

Modeling and visualizing MINOS1's role in cristae junction formation is challenging due to the complex three-dimensional architecture and dynamic nature of these structures. Here are methodological approaches to address this challenge:

• Advanced Microscopy Techniques:

  • Super-resolution microscopy: Techniques such as STED, STORM, or PALM can achieve resolution beyond the diffraction limit (down to ~20 nm), allowing visualization of MICOS components at cristae junctions

  • Correlative light and electron microscopy (CLEM): Combines fluorescence localization of tagged MINOS1 with ultrastructural context from electron microscopy

  • Cryo-electron tomography: Provides 3D visualization of mitochondrial membranes in a near-native state, revealing the organization of cristae junctions where MINOS1 functions

  • Expansion microscopy: Physical expansion of specimens can improve resolution of conventional microscopes for visualizing MICOS complex organization

• Dynamic Visualization Approaches:

  • Live-cell imaging with fluorescently tagged MINOS1 to track its dynamics during mitochondrial fission, fusion, or stress responses

  • Photoactivatable or photoconvertible fluorescent tags to track subpopulations of MINOS1 molecules over time

  • FRAP (Fluorescence Recovery After Photobleaching) to measure MINOS1 mobility and exchange rates at cristae junctions

• Computational Modeling:

  • Molecular dynamics simulations of MINOS1 and its interaction partners within a membrane environment

  • Coarse-grained models of cristae junction formation incorporating known constraints from experimental data

  • Systems biology approaches modeling the assembly and disassembly kinetics of MICOS complexes

  • Integration of structural data from various sources (X-ray crystallography, NMR, cryo-EM) to build comprehensive models

• In Vitro Reconstitution Systems:

  • Synthetic membrane systems (liposomes, nanodiscs) incorporating purified MINOS1 and other MICOS components

  • Microfluidic platforms allowing controlled manipulation of membrane properties

  • High-speed atomic force microscopy to directly visualize membrane remodeling by MICOS components

  • In vitro cryo-EM of reconstituted systems to capture intermediate states of cristae junction formation

• Genetic Engineering Approaches for Visualization:

  • Split-GFP complementation to visualize specific protein-protein interactions within the MICOS complex

  • FRET sensors to detect conformational changes or interactions between MICOS components

  • Tetracysteine tags and biarsenical dyes for pulse-chase imaging of MINOS1 populations

  • Proximity labeling (APEX2, BioID) to map the local environment of MINOS1 at cristae junctions

By combining these methodological approaches, researchers can develop sophisticated models of how MINOS1 contributes to cristae junction formation and maintenance. Such models would integrate structural, dynamic, and functional data to provide a comprehensive understanding of this critical aspect of mitochondrial inner membrane organization.

What approaches can overcome the difficulties in generating viable MINOS1 knockout models for functional studies?

Creating viable MINOS1 knockout models presents challenges due to the protein's essential role in mitochondrial function. Here are methodological approaches to overcome these difficulties:

By employing these methodological strategies, researchers can overcome the challenges associated with generating viable MINOS1 knockout models, enabling detailed functional studies of this essential mitochondrial protein while minimizing confounding effects from complete loss of viability.