Recombinant Dictyostelium citrinum ATP synthase subunit 9, mitochondrial (atp9)

What is the basic structure and function of Dictyostelium citrinum ATP synthase subunit 9?

Atp9 (also called subunit 9 or subunit c) is an essential component of the F₀ domain of mitochondrial ATP synthase. This extremely hydrophobic protein contains two transmembrane segments and forms a ring structure composed of multiple identical subunits (typically 10 in yeast) that functions as part of the proton-translocating domain. During ATP synthesis, the subunit 9 ring rotates as protons are transported across the mitochondrial inner membrane, resulting in conformational changes that drive ATP production by the catalytic head (F₁) of the ATP synthase .

How does the genomic location of ATP9 differ among eukaryotes?

In eukaryotes, ATP9 can be encoded either by the mitochondrial genome or the nuclear genome, representing a fascinating case of gene transfer during evolution. In Saccharomyces cerevisiae, ATP9 is encoded by the mitochondrial genome, while in filamentous fungi like Podospora anserina and many animals, ATP9 has been transferred to the nuclear genome . In Trypanosoma brucei, the ATP9 gene has been identified in the nuclear genome with a putative mitochondrial import sequence at the N-terminus . Comparative analysis of social amoebae mitochondrial genomes shows that Dictyostelium citrinum, like other Dictyostelium species, has retained ATP9 in its mitochondrial genome .

What amino acid sequences are covered by the tRNAs in Dictyostelium citrinum mitochondrial genome?

The mitochondrial genome of Dictyostelium citrinum encodes tRNAs that cover 15 amino acids, which is the same coverage as observed in D. discoideum and P. pallidum, but more extensive than the 13 amino acids covered in Acanthamoeba castellanii. This indicates that all these organisms must import additional tRNAs from the cytosol to support complete mitochondrial protein synthesis .

What expression systems are most suitable for recombinant production of Dictyostelium citrinum Atp9?

For recombinant expression of Dictyostelium citrinum Atp9, researchers should consider specialized expression systems capable of handling highly hydrophobic membrane proteins. Based on successful approaches with other ATP9 genes, viable strategies include:

• Yeast expression systems: The study by Giraud et al. demonstrated successful expression of P. anserina ATP9 genes in S. cerevisiae using both centromeric (pCM189, CEN) and multicopy (pCM190, 2μ) plasmids with a Tet-off (doxycycline-repressible) promoter .

• Codon optimization: When expressing D. citrinum ATP9 in heterologous systems, codon optimization for the host organism is essential, as demonstrated in the successful expression of P. anserina Atp9 genes in S. cerevisiae .

• Addition of appropriate mitochondrial targeting sequences (MTS): An effective MTS, such as that from P. anserina ATP9-7 precursor, is crucial for proper import into mitochondria .

What specific challenges arise when expressing highly hydrophobic proteins like Atp9, and how can they be addressed?

Expressing extremely hydrophobic proteins like Atp9 presents several challenges:

• Import difficulties: Highly hydrophobic proteins can be challenging to import into mitochondria. Giraud et al. found that when attempting to express a recoded yeast ATP9 gene from the nucleus (yAtp9-Nuc), the protein could not cross the mitochondrial inner membrane and was detected only in very small amounts exclusively in mitochondria .

• Protein degradation: Unimported hydrophobic proteins are often rapidly degraded by proteases like the i-AAA protease (Yme1p). This was confirmed by expressing yAtp9-Nuc in a Δyme1 strain, which showed increased protein accumulation .

• Solutions: Successful expression strategies include:

  • Reducing hydrophobicity of the first transmembrane segment to facilitate import

  • Using naturally nuclear versions from other organisms that have evolved mechanisms to overcome these challenges

  • Employing appropriate targeting sequences optimized for the specific protein

How can researchers verify proper mitochondrial localization and assembly of recombinant Atp9?

Verification of proper mitochondrial localization and assembly of recombinant Atp9 requires multiple complementary approaches:

• Subcellular fractionation and Western blotting: Isolate mitochondria and use antibodies against Atp9 to detect the protein specifically in mitochondrial fractions. Compare the size of detected proteins with native Atp9 to determine proper processing of targeting sequences .

• Respiratory function assays: Measure oxygen consumption rates in mitochondria (state 3 with NADH as an electron donor) to assess functional incorporation of recombinant Atp9 into the ATP synthase complex. In the study by Giraud et al., strains expressing P. anserina ATP9 genes showed oxygen consumption rates of 40-80% compared to wild-type .

• Blue Native PAGE analysis: This technique allows visualization of assembled ATP synthase complexes to determine whether recombinant Atp9 has been incorporated into functional complexes .

• Growth assays on respiratory substrates: The ability of cells expressing recombinant Atp9 to grow on non-fermentable carbon sources (like glycerol) provides evidence of functional oxidative phosphorylation .

What approaches can be used to study the assembly-dependent translation regulation of Atp9?

Recent research has revealed that translation of ATP synthase components, including Atp9, can be regulated by assembly feedback mechanisms. To study this phenomenon:

• Create specific assembly mutants: Generate strains with mutations affecting specific steps in ATP synthase assembly to observe effects on Atp9 translation.

• Pulse-chase experiments: These can determine if translation rates of Atp9 are enhanced in mutant strains with specific defects in assembly.

• Analyze assembly intermediates: Identify and characterize assembly intermediates that interact with newly synthesized Atp9.

• Investigate cis-regulatory sequences: Examine the role of sequences within the mitochondrial genome that control gene expression in response to assembly status .

Research by Rak et al. suggests that Atp9 translation may be part of an assembly-dependent feedback loop, contradicting the generally accepted view that the subunit 9 ring forms separately and independently of other ATP synthase components .

How does D. citrinum Atp9 compare structurally and functionally with Atp9 from other organisms?

Comparative analysis shows several key points:

• Sequence homology: Among social amoebae, D. citrinum Atp9 shares significant sequence similarity with D. discoideum Atp9. The mitochondrial genomes of these species show similar patterns of codon usage, with D. citrinum using TAA as a stop codon 36 times, TAG 6 times, and TGA once, comparable to the pattern in D. discoideum .

• Hydrophobicity profiles: The extreme hydrophobicity of Atp9 is a conserved feature across species, but subtle differences exist. In organisms where ATP9 has been transferred to the nucleus (like P. anserina), there appears to be reduced hydrophobicity in the first transmembrane segment, which facilitates mitochondrial import .

• Functional complementation: When testing cross-species compatibility, studies have shown that naturally nuclear versions of Atp9 (like those from P. anserina) can functionally complement Atp9 deficiency in other species (like S. cerevisiae), though not with 100% efficiency. This suggests structural and functional conservation with species-specific adaptations .

What can comparative studies of ATP9 gene location across different Dictyostelium species tell us about mitochondrial genome evolution?

Comparative analysis of ATP9 gene location across Dictyostelium species offers valuable insights:

• Conservation pattern: ATP9 is retained in the mitochondrial genome of multiple Dictyostelium species including D. discoideum, D. citrinum, and D. fasciculatum, suggesting evolutionary conservation of this arrangement within this clade .

• Genome rearrangements: Analysis of mitochondrial genome organization in D. discoideum, D. citrinum, P. pallidum, and D. fasciculatum revealed specific rearrangements. For example, the arrangement of segments containing ATP9 differs between some species, with a rearrangement occurring after the divergence of group 4 Dictyostelia .

• Transcriptional organization: In D. discoideum, transcription of the entire mitochondrial genome, including ATP9, is initiated at a single site, generating a large polycistronic transcript that is efficiently processed. This contrasts with the multiple transcription initiation sites found in many other eukaryotes and represents the first report of a protist mitochondrial DNA that, although much larger than metazoan counterparts, is transcribed from a single initiation site .

What are the main challenges in achieving functional allotopic expression of ATP9, and how can they be overcome?

Allotopic expression (relocating a mitochondrial gene to the nucleus) of ATP9 presents several challenges:

• Hydrophobicity barrier: The extreme hydrophobicity of Atp9 makes it difficult to import into mitochondria when expressed from the nucleus. Previous attempts with yeast Atp9p showed the protein could not cross the mitochondrial inner membrane efficiently.

• Protein degradation: Unimported or improperly folded recombinant Atp9 is rapidly degraded by quality control proteases like the i-AAA protease (Yme1p).

• Successful strategies demonstrated by Giraud et al. include:

  • Using naturally nuclear versions from other organisms (like P. anserina) that have evolved solutions to these problems

  • Reducing the hydrophobicity of the first transmembrane segment to facilitate import

  • Utilizing appropriate mitochondrial targeting sequences

  • Optimizing codon usage for expression in the nuclear genetic context

  • Testing both centromeric and multicopy plasmids to find optimal expression levels

How does the expression of nuclear-encoded Atp9 affect cellular morphology and stress responses?

Nuclear expression of ATP9, while supporting oxidative phosphorylation, can disrupt cellular homeostasis:

• Cellular morphology alterations: Cells expressing nuclear versions of ATP9 may exhibit altered cellular morphology compared to cells with mitochondrially-encoded ATP9.

• Stress response activation: Giraud et al. observed that nuclear expression of ATP9 activated the heat shock response in yeast cells, indicating cellular stress despite the maintenance of near-normal oxidative phosphorylation function.

• Import and assembly challenges: Even with successful complementation, cells expressing nuclear-encoded Atp9 showed evidence of difficulty in importing and properly assembling the protein into functional ATP synthase complexes .

These findings highlight the complex cellular adaptations required for successful mitochondrial gene transfer and suggest why ATP9 remained in the mitochondrial genome in many lineages while being transferred to the nucleus only in some organisms during evolution.

Data Table: Codon Usage in Mitochondrial Genomes of Social Amoebae