Recombinant Saccharomyces cerevisiae ATP synthase subunit 9, mitochondrial (OLI1)

What is the structural organization of ATP synthase subunit 9 in S. cerevisiae mitochondria?

ATP synthase subunit 9 (Atp9p) is a core component of the F0 domain in the mitochondrial F1F0-ATP synthase complex. Within the fully assembled enzyme, Atp9p forms an oligomeric ring structure composed of 10 identical subunits, referred to as the 9₁₀-ring. This ring is embedded in the inner mitochondrial membrane and forms part of the proton channel along with subunit 6 (Atp6p) . During proton translocation, the 9₁₀-ring rotates, which induces conformational changes in the F1 domain that catalyze ATP synthesis . The functional mitochondrial ATP synthase in yeast is an assembly of 28 subunits of 17 different types, with only three (subunits 6, 8, and 9) encoded by mitochondrial genes and the remaining 14 encoded by nuclear genes .

How does the stoichiometry of subunit 9 differ from other mitochondrially-encoded ATP synthase components?

Mitochondria contain approximately 10 times more subunit 9 than subunits 6 or 8 . This differential expression is likely attributable to two main factors:

• Higher concentration of ATP9 mRNA relative to the ATP8/6 mRNAs in the mitochondrial matrix

• Distinct translational regulation mechanisms, as each gene depends on a separate translational activator

This stoichiometric imbalance reflects the structural requirements of the ATP synthase, where ten subunit 9 proteins must assemble to form the functional 9₁₀-ring, while only one copy each of subunits 6 and 8 are incorporated into the mature complex.

How is the ATP9/OLI1 gene organized and transcriptionally regulated in yeast mitochondria?

The ATP9/OLI1 gene in yeast mitochondria is part of a polycistronic transcription unit. It is co-transcribed with a serine tRNA gene (tRNA^ser) and the VAR1 gene (encoding a mitochondrial ribosomal protein) . Transcription initiates from two closely spaced promoters corresponding to conserved nonanucleotide sequences:

• The primary transcription start site is located 0.63 kb upstream of the ATP9 initiation codon

• A secondary site is positioned 0.55 kb upstream of the ATP9 start codon

The resulting polycistronic transcript undergoes post-transcriptional processing through multiple endonucleolytic cleavages to generate the mature tRNA^ser and the ATP9 and VAR1 mRNAs. The major ATP9 mRNA is approximately 0.9 kb with a remarkably long 5′ untranslated region of 0.63 kb . Processing of the 3' ends occurs near a common seven-nucleotide sequence (5'-ATTCTTA-3'), which is also found in other mitochondrial genes and may function as a signal for transcription termination or RNA processing .

What mechanisms regulate translation of ATP9/OLI1 and how do they differ from other mitochondrially-encoded ATP synthase subunits?

Translation of ATP9/OLI1 is regulated through distinct mechanisms compared to ATP6 and ATP8. Recent research has revealed complex regulatory networks:

• Assembly-dependent feedback: Translation of subunits 6 and 9 can be enhanced in mutant strains with specific defects in the assembly of these proteins. This indicates that assembly intermediates interact with these proteins within the final ATP synthase complex .

• Cis-regulatory sequences: Expression is controlled by specific sequences within the mitochondrial genome, particularly in the extensive 5' UTR of the ATP9 transcript .

• Translational activators: Each mitochondrially-encoded ATP synthase subunit has dedicated nuclear-encoded translational activators. For ATP9, these factors interact with the 5' UTR to promote translation initiation .

• F1-dependent translation: Unlike ATP9, translation of ATP6 and ATP8 is strictly dependent on the presence of the F1 ATPase module, providing a mechanism for coordinated production of ATP synthase components .

These regulatory differences ensure proper stoichiometric production of ATP synthase subunits despite their dual genetic origin (nuclear and mitochondrial).

What is the current model for ATP synthase assembly in yeast, and what role does subunit 9 play in this process?

The assembly of ATP synthase in S. cerevisiae follows a modular pathway rather than a single linear process as previously thought. Recent research using pulse-labeling and pulse-chase experiments has identified two separate but coordinately regulated assembly pathways that converge at the final stage .

The current model includes these key steps and intermediates:

• F1/Atp9p ring intermediate: One assembly pathway produces a subcomplex consisting of the F1 ATPase module bound to the Atp9p ring. This interaction is rapid and occurs independently of Atp6p and Atp8p .

• Atp6p/Atp8p/stator intermediate: The second pathway produces a complex containing Atp6p, Atp8p, at least two stator subunits, and the Atp10p chaperone .

• Terminal assembly: These two subcomplexes unite in the final step of assembly to form the complete F1F0-ATP synthase .

This model contradicts earlier views that the 9₁₀-ring forms completely independently. Instead, the assembly process appears designed to prevent premature formation of the proton-conductive channel (at the interface of Atp6p and the Atp9p ring) until a fully coupled ATP synthase is assembled, thereby preventing proton leakage across the membrane .

How do assembly factors and chaperones facilitate the incorporation of subunit 9 into the ATP synthase complex?

Multiple nuclear-encoded factors facilitate the assembly of subunit 9 into the ATP synthase complex:

• INA complex (INAC): This recently identified complex physically associates with two distinct ATP synthase subassemblies: the Atp9p ring and the module containing Atp6p, Atp8p, F1, and the peripheral stalk. INAC appears to function in the terminal step of F1F0-ATP synthase assembly, specifically mediating the association of the Atp9p ring with Atp6p .

• ATP synthase-specific assembly factors: Several nuclear-encoded proteins specifically promote the assembly of the F0 sector, including factors that facilitate the formation and stability of the Atp9p ring .

• Translational activators: These proteins not only promote translation of ATP9 mRNA but may also coordinate the co-translational insertion of Atp9p into the inner mitochondrial membrane .

• F1 module: The F1 ATPase interacts with newly synthesized Atp9p, potentially stabilizing the ring structure and facilitating its proper assembly .

Defects in these assembly factors lead to respiratory deficiencies, underscoring their essential role in ATP synthase biogenesis and mitochondrial function.

What are the most effective methods for studying ATP9/OLI1 expression and Atp9p assembly in yeast mitochondria?

Several complementary approaches have proven valuable for investigating ATP9/OLI1 expression and Atp9p assembly:

• In organello pulse labeling and pulse-chase experiments: These techniques enable tracking of newly synthesized mitochondrially-encoded proteins, including Atp9p, and identification of assembly intermediates .

  Methodology:

  • Isolate intact mitochondria from S. cerevisiae

  • Pulse-label with [35S]methionine/cysteine to radiolabel newly synthesized mitochondrial proteins

  • Chase with cold methionine/cysteine for various time periods

  • Extract with digitonin and analyze complexes by native gel electrophoresis or immunoprecipitation

• S1 nuclease mapping and RNA hybridization: These approaches are used to characterize ATP9 transcripts, including mapping the 5' and 3' ends and quantifying transcript levels .

• Immunoprecipitation with subunit-specific antibodies: This technique can identify interacting partners and assembly intermediates containing Atp9p .

  Example protocol:

  • Extract mitochondrial proteins with digitonin

  • Treat with antibodies against specific ATP synthase components (e.g., F1)

  • Analyze precipitates by SDS-PAGE and autoradiography to visualize labeled subunits

• Nuclear expression of mitochondrially-encoded subunits: Engineering nuclear-encoded versions of Atp9p (Atp9-nuc) allows investigation of assembly defects without interference from translation defects .

What genetic approaches can be employed to investigate ATP9/OLI1 function in vivo?

Several genetic strategies have proven valuable for investigating ATP9/OLI1 function:

• Respiratory-deficient mutants: Analysis of yeast strains with respiratory deficiencies due to mutations affecting ATP synthase biogenesis has been instrumental in identifying nuclear gene products involved in ATP9 expression and Atp9p assembly .

• Specific assembly mutants: Strains with targeted disruptions in assembly factors such as INAC components display ATP synthase biogenesis defects, including dissociation of the F1 module .

• Site-directed mutagenesis: Introducing specific mutations in ATP9/OLI1 can provide insights into structure-function relationships and assembly requirements.

How does the assembly-dependent translational regulation of ATP9/OLI1 challenge existing models of mitochondrial gene expression?

Recent findings about assembly-dependent translational regulation of ATP9/OLI1 significantly challenge previously established models:

• The discovery that translation of subunit 9 is enhanced in mutant strains with specific assembly defects contradicts the view that mitochondrial gene expression is primarily regulated at the transcriptional level .

• The finding that Atp9p forms part of an assembly-dependent feedback loop suggests a complex regulatory network coordinating mitochondrial and nuclear gene expression .

• Evidence that the Atp9p ring does not form completely independently of other ATP synthase components contradicts the generally accepted assembly model where the 9₁₀-ring was thought to assemble separately .

These discoveries suggest a more integrated and dynamic regulation of mitochondrial gene expression than previously recognized, where the assembly state of protein complexes directly influences the translation of their components. This represents a paradigm shift in our understanding of organellar gene expression coordination.

What are the implications of ATP synthase modular assembly for understanding mitochondrial diseases associated with ATP synthase deficiency?

The modular assembly of ATP synthase has significant implications for understanding mitochondrial diseases:

• Multiple vulnerable points: The discovery of two convergent assembly pathways suggests multiple points where defects can arise, potentially explaining the diverse clinical presentations of ATP synthase deficiencies.

• Coordinated regulation: The synchronized assembly of nuclear and mitochondrially-encoded components requires precise coordination. Disruption of this regulation could contribute to disease pathogenesis even when individual subunits are structurally normal.

• Proton leak prevention: The segregation of Atp6p from the Atp9p ring until the final assembly stage prevents premature formation of the proton channel, avoiding proton leakage across the membrane . Defects in this orchestrated assembly could lead to mitochondrial uncoupling and energy deficit.

• Therapeutic targets: Understanding the roles of assembly factors like the INA complex provides potential targets for therapeutic interventions in mitochondrial diseases associated with ATP synthase dysfunction.

What contradictions exist in the current data regarding Atp9p ring formation and integration into the ATP synthase complex?

Several notable contradictions and unresolved questions exist in the current understanding of Atp9p ring formation:

• Independent vs. dependent assembly: While earlier models suggested that the Atp9p ring forms completely independently, recent evidence indicates that its proper assembly involves interactions with the F1 module and potentially other factors .

• Assembly factor requirements: There are conflicting data regarding which specific assembly factors are absolutely required for Atp9p ring formation versus those needed for its integration into the mature ATP synthase.

• Temporal sequence: The precise timing of Atp9p oligomerization relative to its interaction with F1 remains unclear, with some data suggesting these processes may occur simultaneously rather than sequentially .

• Regulatory mechanisms: While evidence suggests assembly-dependent feedback regulation of Atp9p translation, the molecular mechanisms and signaling pathways involved in this process remain poorly defined .

These contradictions highlight the need for further research using advanced structural and biochemical approaches to fully elucidate the complex process of ATP synthase assembly in yeast mitochondria.