Recombinant Saccharomyces cerevisiae ATP synthase subunit K, mitochondrial (ATP19)

What is the role of ATP19 in the yeast mitochondrial ATP synthase complex?

ATP19 (subunit k) functions as one of the supernumerary subunits of the ATP synthase complex in Saccharomyces cerevisiae. It is present in both monomeric and dimeric forms of the complex . ATP19's primary function appears to be structural stabilization of the ATP synthase complex, particularly supporting the formation and maintenance of ATP synthase dimers. Structurally, ATP19 attaches to the complex via its N-terminal helix, which fits into a V-shaped groove formed by helices 4 and 5 of subunit ATP6/a . While ATP19 deletion does not completely inhibit ATP synthase function, it reduces efficiency and stability, particularly under stressful conditions.

How is ATP19 integrated into the assembly pathway of ATP synthase?

ATP19 is incorporated during the modular assembly of the mitochondrial ATP synthase. Research indicates that ATP synthase assembly is not a single linear process but rather involves two separate but coordinately regulated pathways that converge at the end stage . ATP19, along with other supernumerary subunits ATP20/g and ATP21/e, appears to "prime" the monomers before dimers are formed . This suggests ATP19 plays a crucial role in the transition from monomeric to dimeric states of ATP synthase, which is essential for proper cristae formation and optimal mitochondrial function.

What are the recommended approaches for investigating ATP19 interactions within the ATP synthase complex?

To study ATP19 interactions within the ATP synthase complex, several complementary methodologies are effective:

• Affinity Purification Mass Spectrometry: Using HA-6xHis-tagged ATP6 bound to Ni-NTA agarose to pull down the full ATP synthase complex, followed by elution, SDS-PAGE separation, and mass spectrometry analysis. This approach has successfully identified ATP19 along with other ATP synthase components .

• Blue Native PAGE (BN-PAGE): Extracting ATP synthase monomers and dimers from isolated mitochondria using 2% digitonin, followed by separation on 3-12% BN-PAGE. This can be combined with a second dimension 16% SDS-PAGE to resolve individual subunits .

• Crosslinking Coupled with Mass Spectrometry: This can provide detailed insights into proximity relationships between ATP19 and neighboring subunits within the complex.

• Yeast Two-Hybrid or Split-Ubiquitin Assays: These can be employed to identify direct protein-protein interactions involving ATP19.

Each of these methods offers unique advantages for understanding ATP19's structural relationships and functional interactions within the ATP synthase complex.

What methods can be used to assess the impact of ATP19 deletion on ATP synthase function?

Assessment of ATP19's functional impact requires multiple complementary approaches:

• Respiratory Growth Assays: Testing growth on non-fermentable carbon sources (e.g., glycerol or ethanol) with and without ATP synthase inhibitors like oligomycin. For example, Δatp19 strains show reduced growth at 1 μg/ml oligomycin at both 28°C and 36°C, revealing a role in ATP synthase stability .

• Oxygen Consumption Measurements: Measuring oxygen consumption in isolated mitochondria with NADH as an electron donor under various conditions:

  • Basal (state 4) respiration

  • Phosphorylating (state 3) conditions after ADP addition

  • Uncoupled (maximal) respiration after CCCP addition

• ATP Synthesis Rate Measurement: Quantifying ATP production by ATP synthase during state 3 respiration, with and without oligomycin inhibition. Δatp19 shows approximately 25% decrease in ATP synthesis rates .

• ATP Hydrolysis Assays: Measuring the rate of ATP hydrolysis in non-osmotically protected mitochondria at pH 8.4 (to avoid IF1 inhibitor binding) .

• BN-PAGE Analysis: Examining the ratio of ATP synthase dimers to monomers extracted from mitochondria lacking ATP19 .

These methodologies collectively provide a comprehensive assessment of how ATP19 affects ATP synthase structure and function.

What expression systems are most effective for producing recombinant ATP19 for structural studies?

For structural studies of recombinant ATP19, several expression systems can be employed with specific considerations:

• E. coli Expression Systems:

  • BL21(DE3) strains with pET vectors allow for high-yield expression

  • Fusion tags (His6, GST, or MBP) facilitate purification and can enhance solubility

  • Codon optimization for E. coli may be necessary given yeast codon bias

  • Expression at lower temperatures (16-18°C) can improve proper folding

• Yeast Expression Systems:

  • S. cerevisiae or Pichia pastoris systems provide eukaryotic post-translational modifications

  • They offer proper folding environment similar to the native context

  • Can be designed with inducible promoters (GAL1 for S. cerevisiae; AOX1 for P. pastoris)

• Cell-Free Expression Systems:

  • Useful for rapid screening of constructs

  • Can incorporate isotopic labeling for NMR studies

  • Allow expression of potentially toxic proteins

For structural determination, recombinant ATP19 should be purified under conditions that maintain its native conformation, possibly including appropriate detergents or lipid environments that mimic the mitochondrial membrane.

How does ATP19 contribute to the formation of ATP synthase dimers?

ATP19 plays a critical role in the formation and stabilization of ATP synthase dimers. Research using Blue Native PAGE has demonstrated that mitochondria lacking ATP19 extract fewer dimeric forms of ATP synthase compared to wild-type, confirming ATP19's role in dimer stabilization . The molecular basis for this stabilization appears to be related to ATP19's structural position within the complex.

Structural analysis reveals that ATP19 attaches to the ATP synthase complex with its N-terminal helix in a V-shaped groove formed by helix 4 and 5 of subunit ATP6/a . This positioning likely provides stability to the interface regions involved in dimer formation. Additionally, ATP19, along with ATP20 and ATP21 subunits, has been shown to "prime" the monomers before dimers are formed , suggesting a sequential assembly process where ATP19 facilitates the transition from monomeric to dimeric states.

The dimerization of ATP synthase is physiologically significant as it contributes to proper cristae formation and optimal mitochondrial function. ATP19's role in this process represents an important regulatory mechanism for ensuring efficient energy production in mitochondria.

What is known about the temporal sequence of ATP19 incorporation during ATP synthase assembly?

The assembly of mitochondrial ATP synthase involves a complex, modular process rather than a single linear pathway. Research indicates that ATP synthase assembly involves two separate but coordinately regulated pathways that converge at the end stage .

Within this framework, evidence suggests that ATP19 is incorporated relatively late in the assembly process. The formation of ATP synthase involves multiple assembly intermediates, with the mitochondrially encoded ATP6, ATP8, and ATP9 subunits segregating into two different assembly pathways:

• One intermediate consists of ATP6, ATP8, at least two stator subunits, and the ATP10 chaperone .

• A second intermediate contains the F1 ATPase and the ATP9 ring .

ATP19, as a supernumerary subunit, appears to be incorporated after these major intermediates have formed, likely during the process that converts monomeric complexes to dimeric forms. This is supported by findings that ATP19, along with ATP20 and ATP21 subunits, "primes" the monomers before the dimers are formed , suggesting a specific temporal sequence in the final stages of ATP synthase assembly.

What structural features of ATP19 are critical for its function in ATP synthase?

Several structural features of ATP19 appear critical for its proper function within the ATP synthase complex:

Understanding these structural features provides insights into how ATP19 contributes to ATP synthase assembly, stability, and function, and offers potential targets for mutagenesis studies to further elucidate its precise role.

How does ATP19 interact with other supernumerary subunits like ATP20 and ATP21?

ATP19 functions in concert with other supernumerary subunits, particularly ATP20/g and ATP21/e, within the ATP synthase complex. All three of these subunits have been identified in both monomeric and dimeric forms of ATP synthase . Research suggests that ATP19, ATP20, and ATP21 work together to "prime" the monomers before dimers are formed , indicating a coordinated function in the transition from monomeric to dimeric states.

Further research using techniques such as cross-linking mass spectrometry or cryo-electron microscopy would be valuable for mapping the precise interaction networks between ATP19, ATP20, and ATP21, and understanding how these interactions collectively contribute to ATP synthase assembly and stability.

What is the relationship between ATP19 and the newly identified Mco10 protein?

Recent research has identified an intriguing relationship between ATP19 and a previously uncharacterized protein called Mco10 (Yor020W-A). Both proteins appear to interact with the ATP synthase complex, but with distinct functional roles:

• Structural Similarities: Based on the conservation of N-terminal fragments and structural analysis, Mco10 and ATP19 might occupy similar positions in the ATP synthase F₀ domain . Computational modeling using AlphaFold2 predicted structures showed that Mco10 can accommodate very well in the same binding pocket as ATP19, with minimal steric hindrance with neighboring subunits ATP18/i, ATP6/a, and the c-ring .

• Functional Differences: Despite structural similarities, Mco10 and ATP19 show different functional profiles:

  • ATP19 deletion significantly reduces growth in the presence of oligomycin

  • Mco10 deletion does not affect respiratory growth under similar conditions

  • Interestingly, Mco10 deletion can rescue the growth defect of ATP19 deletion at 28°C to some extent

  • In the presence of oligomycin, which completely blocks ATP synthase in Δatp19, the ATP synthesis rate was twice higher in Δmco10 compared to wild type mitochondria

• Complex Localization: While both proteins were identified in ATP synthase monomers and dimers by mass spectrometry, Mco10 appears more abundant in the monomeric form based on western blot analysis .

These findings suggest that Mco10 and ATP19 may have complementary or possibly antagonistic roles in regulating ATP synthase function, particularly in relation to oligomycin sensitivity and the transition between monomeric and dimeric states.

How do mutations in ATP19 affect interactions with the c-ring and proton transport?

The positioning of ATP19 in proximity to both the proton half-channel and the oligomycin binding site in the c-ring suggests that ATP19 may influence proton transport and sensitivity to inhibitors. Several key observations support this relationship:

• Structural Proximity: ATP19 attaches to the complex via its N-terminal helix in a V-shaped groove formed by helices 4 and 5 of ATP6/a , positioning it near critical functional elements of the proton transport machinery.

• Oligomycin Sensitivity: Deletion of ATP19 (Δatp19) significantly reduces growth in the presence of suboptimal concentrations of oligomycin . Since oligomycin binds to the c-ring and blocks proton transport, this suggests that ATP19 influences the conformational state or accessibility of the oligomycin binding site.

• ATP Synthesis Efficiency: ATP19 deletion leads to approximately 25% decrease in ATP synthesis rates , indicating a partial uncoupling or reduced efficiency of the proton transport mechanism.

These findings suggest that ATP19 may play a role in maintaining the optimal structural arrangement of the c-ring and proton channel components, ensuring efficient coupling between proton transport and ATP synthesis. Mutations in ATP19 likely disrupt these interactions, leading to altered sensitivity to inhibitors and reduced efficiency of energy conversion.

Detailed mutagenesis studies targeting specific regions of ATP19 that interface with the c-ring or proton channel components would provide valuable insights into the precise molecular mechanisms by which ATP19 influences these critical functions.

How conserved is ATP19 across different yeast species and what does this reveal about its function?

Phylogenetic analysis of ATP19 across fungal species reveals important insights about its evolutionary conservation and functional significance:

• Conservation Pattern: Many related yeast species maintain both ATP19 and Mco10 proteins , suggesting that both proteins provide selective advantages that have been maintained through evolution. The conservation of ATP19 across multiple fungal species indicates that its function is fundamental to mitochondrial ATP synthase across this taxonomic group.

• N-terminal Conservation: The N-terminal fragments of ATP19 show particular conservation across yeast species . This conservation pattern aligns with structural data showing that ATP19 attaches to the ATP synthase complex via its N-terminal helix , suggesting that this interaction interface is functionally critical and therefore evolutionarily conserved.

• Functional Implications: The conservation of ATP19 across fungal species, particularly in regions that interface with other ATP synthase subunits, suggests that its role in stabilizing ATP synthase structure, especially dimeric forms, represents a conserved mechanism for regulating ATP synthase efficiency and organization within mitochondria.

• Species-Specific Variations: While the core functional domains show conservation, species-specific variations in ATP19 sequence and structure likely reflect adaptations to different metabolic requirements or environmental conditions across yeast species.

This evolutionary conservation pattern provides strong evidence for ATP19's fundamental importance in ATP synthase function across fungi and offers insights into which structural elements are most critical for its function.

What functional equivalents of ATP19 exist in higher eukaryotes?

• Sequence Homology: Direct sequence homology searches may identify proteins with similar primary structures in metazoan mitochondrial ATP synthase complexes.

• Structural Homology: Even with limited sequence conservation, structural homology may reveal proteins that occupy similar positions within the ATP synthase complex of higher eukaryotes. The search results mention comparison of yeast and human ATP synthase interactomes , suggesting potential for identifying functional equivalents.

• Functional Conservation: Proteins that perform similar functions in stabilizing ATP synthase dimers in higher eukaryotes may represent functional equivalents even with limited sequence similarity.

• Evolutionary Analysis: Phylogenetic profiling across diverse eukaryotic lineages can help trace the evolutionary trajectory of ATP19-like proteins and identify potential orthologs or functional replacements in different taxonomic groups.

Understanding these equivalents in higher eukaryotes has significant implications for biomedical research, as mitochondrial ATP synthase dysfunction is implicated in various human diseases. Identifying the human counterparts of yeast ATP19 could provide insights into disease mechanisms and potential therapeutic targets.

What can we learn from comparing recombinant ATP19 expression in different host systems?

Comparing the expression of recombinant ATP19 across different host systems provides valuable insights into protein folding, function, and host-specific factors that influence ATP synthase assembly:

• Protein Folding and Stability: Different expression hosts may yield ATP19 with varying conformational properties. Comparing structural characteristics of ATP19 expressed in prokaryotic systems (E. coli) versus eukaryotic systems (yeast, insect cells) can reveal requirements for proper folding.

• Post-translational Modifications: Expression in different systems may result in varying post-translational modifications that could impact ATP19 function. Comparing these modifications can reveal their functional significance.

• Assembly Compatibility: Testing whether recombinant ATP19 expressed in heterologous systems can complement ATP19 deletion in yeast provides insights into structural requirements for functional integration into the ATP synthase complex.

• Interaction Networks: Comparing the protein-protein interaction networks of recombinant ATP19 expressed in different systems can reveal host-specific factors that influence ATP synthase assembly.

• Cross-species Compatibility: Similar to studies with ATP9, where a nucleus-encoded version from Podospora anserina (ATP9-nuc) could partially complement an ATP9 deletion in yeast , cross-species complementation studies with ATP19 from different organisms could reveal evolutionary constraints and functional conservation.

This comparative approach not only enhances our understanding of ATP19 biology but also provides practical insights for optimizing recombinant protein production for structural and functional studies.

What are the major technical challenges in studying ATP19 and how can they be overcome?

Studying ATP19 presents several technical challenges that require specific methodological solutions:

• Size Limitations:

  • Challenge: ATP19 is a small protein, making it difficult to detect and analyze with standard techniques.

  • Solution: Use specialized gel systems optimized for small proteins, such as Tricine-SDS-PAGE or 16% SDS-PAGE, as used in the reported studies . Mass spectrometry approaches specifically optimized for small proteins can also enhance detection sensitivity.

• Mitochondrial Environment Replication:

  • Challenge: Maintaining the native lipid environment crucial for ATP19 function in in vitro studies.

  • Solution: Incorporate appropriate lipid compositions in purification and analysis buffers, or use nanodiscs/liposomes to reconstitute a membrane-like environment for functional studies.

• Protein-Protein Interactions:

  • Challenge: Capturing the transient or weak interactions that ATP19 forms with other ATP synthase subunits.

  • Solution: Employ gentle crosslinking approaches followed by mass spectrometry, or use proximity labeling techniques such as BioID or APEX to map the interaction landscape.

• Genetic Redundancy:

  • Challenge: Potential functional overlap between ATP19 and Mco10 may mask phenotypes in single deletion studies.

  • Solution: Generate and analyze double mutants (such as Δatp19Δmco10) as demonstrated in the research , and employ conditional expression systems to study essential gene combinations.

• Recombinant Expression:

  • Challenge: Achieving proper folding and membrane integration of recombinant ATP19.

  • Solution: Test multiple expression systems, including cell-free systems that incorporate lipids, and optimize conditions specific to membrane proteins.

Addressing these technical challenges requires an integrated approach combining genetic, biochemical, and structural methodologies tailored to the unique properties of ATP19.

How can researchers effectively distinguish between ATP19 and other small ATP synthase subunits?

Distinguishing ATP19 from other small ATP synthase subunits requires multiple complementary approaches:

• 2D Gel Electrophoresis:

  • First dimension: Blue Native PAGE to separate ATP synthase complexes

  • Second dimension: High-resolution SDS-PAGE (16-20%) optimized for small proteins

  • This approach was successfully used in the research to resolve ATP19 and other small subunits

• Mass Spectrometry:

  • Targeted proteomics approaches like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

  • These methods can detect specific peptide signatures unique to ATP19

  • Ultra-high resolution mass spectrometry can distinguish between proteins of similar size

• Immunological Methods:

  • Develop highly specific antibodies against unique epitopes of ATP19

  • Use epitope tagging approaches (HA, FLAG, etc.) for recombinant versions

  • Western blotting with optimized conditions for small proteins

• Genetic Approaches:

  • Create strains with differentially tagged versions of small subunits

  • Use CRISPR-based approaches for endogenous tagging

• Comparative Migration Analysis:

  • Establish a reference map of migration patterns for all small ATP synthase subunits

  • Use known deletion strains as controls to confirm band identities

These approaches, used in combination, can provide reliable discrimination between ATP19 and other small subunits of the ATP synthase complex, enabling accurate analysis of their specific roles and interactions.

What are the most effective methods for measuring the impact of ATP19 on ATP synthase efficiency?

To accurately measure the impact of ATP19 on ATP synthase efficiency, several complementary approaches can be employed:

• ATP Synthesis Rate Measurement:

  • Method: Isolate mitochondria from wild-type and Δatp19 strains and measure ATP production rates under controlled conditions.

  • Implementation: Use luciferin-luciferase assays for real-time ATP quantification. The research showed that ATP19 deletion leads to approximately 25% decrease in ATP synthesis rates .

  • Controls: Include measurements with specific inhibitors like oligomycin to distinguish ATP synthase-specific activity.

• Respiratory Capacity Assessment:

  • Method: Measure oxygen consumption in isolated mitochondria under different respiratory states.

  • Implementation: Use oxygen electrodes or plate-based respirometry to assess:

    • Basal (state 4) respiration

    • Phosphorylating (state 3) conditions after ADP addition

    • Uncoupled (maximal) respiration after CCCP addition

  • Analysis: Calculate respiratory control ratios (state 3/state 4) to assess coupling efficiency.

• Membrane Potential Measurements:

  • Method: Assess the proton gradient across the inner mitochondrial membrane.

  • Implementation: Use potential-sensitive fluorescent dyes like TMRM or JC-1.

  • Relevance: Decreased coupling efficiency in Δatp19 would affect membrane potential maintenance.

• ATP Hydrolysis Assays:

  • Method: Measure the ATP hydrolysis activity with and without inhibitors.

  • Implementation: Perform in non-osmotically protected mitochondria at pH 8.4 to avoid natural inhibitor binding .

  • Analysis: Compare oligomycin-sensitive ATPase activity between wild-type and mutant strains.

• Growth Rate Analysis:

  • Method: Monitor growth on non-fermentable carbon sources under various conditions.

  • Implementation: Test growth with and without ATP synthase inhibitors at different temperatures, as demonstrated in the research where Δatp19 showed sensitivity to suboptimal oligomycin concentrations .

  • Quantification: Use growth curve analysis for precise measurement of growth rates and lag phases.

What are the most promising approaches for resolving the structural basis of ATP19 function?

Several cutting-edge approaches hold promise for elucidating the structural basis of ATP19 function:

• Cryo-Electron Microscopy (Cryo-EM):

  • High-resolution cryo-EM of ATP synthase complexes with and without ATP19

  • Focused refinement on the ATP19 region to maximize resolution

  • Time-resolved cryo-EM to capture different conformational states during ATP synthesis

• Integrative Structural Biology:

  • Combine X-ray crystallography of subcomplexes with cryo-EM of the full complex

  • Integrate structural data with crosslinking mass spectrometry (XL-MS) to define distance constraints

  • Validate structural models using molecular dynamics simulations

• Site-Directed Mutagenesis:

  • Systematic alanine scanning of ATP19 to identify critical residues

  • Introduction of cysteine residues for site-specific crosslinking

  • Creation of chimeric proteins between ATP19 and Mco10 to identify functional domains

• AlphaFold2 and Structural Prediction:

  • Leverage advances in protein structure prediction, as already demonstrated in the research

  • Validate predictions with experimental data

  • Model protein-protein interfaces between ATP19 and other subunits

• Single-Molecule Studies:

  • Use fluorescence resonance energy transfer (FRET) to monitor conformational changes

  • Apply high-speed atomic force microscopy (HS-AFM) to visualize ATP synthase dynamics

  • Develop nanodisk systems to study ATP19 in a membrane environment

These approaches, especially when used in combination, have the potential to provide unprecedented insights into how ATP19 contributes to ATP synthase structure, stability, and function at the molecular level.

How might targeting ATP19 advance our understanding of mitochondrial disorders?

While ATP19 is specific to yeast, understanding its role and identifying functional equivalents in higher eukaryotes could significantly advance our understanding of mitochondrial disorders:

This research direction represents an important bridge between basic science investigations of ATP synthase structure and function and clinical applications in the field of mitochondrial medicine.

What emerging technologies could advance ATP19 research in the next five years?

Several emerging technologies hold particular promise for advancing ATP19 research in the coming years:

• Cryo-Electron Tomography (Cryo-ET):

  • Enables visualization of ATP synthase in its native membrane environment

  • Could reveal how ATP19 contributes to supramolecular organization of ATP synthase complexes in mitochondrial cristae

  • Recent advances in sample preparation and computational methods are improving resolution to near-atomic levels

• AI-Driven Structural Biology:

  • Building on the already utilized AlphaFold2 predictions , next-generation AI tools will enable more accurate modeling of protein complexes and dynamics

  • Could predict effects of mutations and guide experimental design

  • May help identify functional equivalents across species through structural rather than sequence homology

• CRISPR-Based Technologies:

  • Base editing and prime editing allow precise genetic modifications without double-strand breaks

  • Enable rapid creation of sequence variants to test structure-function hypotheses

  • Facilitate high-throughput screening of ATP19 mutations

• Single-Cell Proteomics:

  • Allows examination of cell-to-cell variation in ATP19 expression and ATP synthase assembly

  • Could reveal heterogeneity in mitochondrial function relevant to disease states

  • May identify previously unrecognized regulatory mechanisms

• Microfluidic Platforms:

  • Enable high-throughput analysis of mitochondrial function in various genetic backgrounds

  • Allow precise control of cellular environment for studying ATP19 function under various stresses

  • Can be combined with live-cell imaging for real-time assessment

• Organoid Technologies:

  • Development of "mitochondria-on-a-chip" systems could allow study of ATP19 equivalents in more complex cellular environments

  • Enables testing of hypotheses in human tissue contexts

These technologies, especially when integrated in multidisciplinary approaches, have the potential to significantly accelerate our understanding of ATP19 and its role in ATP synthase function, with implications for both basic science and biomedical applications.