Recombinant Yarrowia lipolytica ATP synthase subunit 9, mitochondrial (ATP9)

What is the molecular structure of Y. lipolytica ATP9?

Y. lipolytica ATP9 is a 76-amino acid mitochondrial protein with the sequence: MQLVLAGKYIGAGLASIGLVGAGIGIAIVFAALINGVSRNPALKGQLFTYSILGFALSEATGLFALMIAFLLLYAV . As a subunit of ATP synthase (also known as Complex V), it forms part of the c-ring in the F₀ domain embedded within the inner mitochondrial membrane. The protein contains highly hydrophobic regions that facilitate its membrane integration, as reflected in its amino acid composition rich in leucine, isoleucine, and alanine residues .

What is the genomic context of ATP9 in Y. lipolytica?

ATP9 is encoded within Y. lipolytica's 47.9 kb mitochondrial genome. The mitochondrial DNA of Y. lipolytica contains genes for three ATP synthase subunits (ATP6, ATP8, and ATP9), all located on the same strand . The mitochondrial genome organization of Y. lipolytica shows similarities to that of Hansenula wingei, with conserved gene order blocks and sequence homology . This genomic architecture contributes to the coordinated expression of mitochondrial respiratory components.

What alternative nomenclature exists for ATP9?

The ATP9 protein is also known by several synonyms including OLI1, ATP synthase subunit 9 (mitochondrial), ATP synthase subunit c, and Lipid-binding protein . In protein databases, it is cataloged under UniProt ID Q37695 . This nomenclature variation reflects both its functional role and historical identification pathways in different research contexts.

What expression systems are effective for recombinant Y. lipolytica ATP9 production?

E. coli has been successfully employed as a heterologous expression system for recombinant Y. lipolytica ATP9 . For optimal expression, the full-length protein (amino acids 1-76) is typically fused with an N-terminal His-tag to facilitate purification . Alternative expression approaches include using Y. lipolytica itself as both the source and expression host, particularly when studying protein interactions within their native context or when post-translational modifications are critical to protein function .

What purification strategies yield high-quality recombinant ATP9?

Purification of ATP9 from Y. lipolytica typically involves:

• Initial solubilization: Using dodecylmaltoside (DDM) to solubilize mitochondrial membranes

• Gradient separation: Centrifugation in digitonin-containing glycerol gradients

• Chromatographic separation: Anion exchange chromatography for final purification

For recombinant His-tagged ATP9 expressed in E. coli, immobilized metal affinity chromatography (IMAC) is the preferred initial purification step, potentially followed by size exclusion chromatography to achieve >90% purity as verified by SDS-PAGE .

What reconstitution methods are recommended for lyophilized ATP9?

For reconstitution of lyophilized ATP9:

• Briefly centrifuge the vial before opening to ensure all content settles at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%) for long-term storage stability

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

This approach maintains protein structure and function while preventing degradation during storage.

How should recombinant ATP9 be optimally stored to maintain activity?

For optimal stability of recombinant Y. lipolytica ATP9:

• Long-term storage: Store at -20°C or preferably -80°C in Tris/PBS-based buffer containing 6% trehalose at pH 8.0

• Working aliquots: Store at 4°C for up to one week to minimize freeze-thaw damage

• Glycerol addition: Incorporate 50% glycerol (final concentration) when preparing storage aliquots

Research indicates that repeated freeze-thaw cycles significantly impact protein integrity, particularly for membrane proteins like ATP9 with extensive hydrophobic regions .

How can ATP9 be utilized in ATP synthase dimer structure studies?

ATP9 plays a critical role in ATP synthase dimer formation and stability. Experimental approaches include:

• Comparative analysis: Purify both monomeric and dimeric ATP synthase complexes from Y. lipolytica to analyze component differences (dimeric complexes contain subunits e, g, and k in addition to standard ATP synthase subunits)

• Electron microscopy: Cryo-EM has successfully resolved ATP synthase dimer structures from Y. lipolytica, revealing the molecular architecture of the dimer interface in the membrane

• Activity assays: Compare ATP hydrolysis activity between monomeric and dimeric forms to correlate structural differences with functional implications

These approaches provide insights into how ATP9 contributes to the higher-order organization of ATP synthase complexes and mitochondrial membrane architecture.

What methods enable investigation of ATP9 interactions with other ATP synthase subunits?

To analyze ATP9 interactions within the ATP synthase complex:

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry analysis identifies interaction sites

• Two-dimensional gel electrophoresis: Resolves ATP synthase complex components while maintaining native interactions

• LC-MS verification: Confirms the presence of specific subunits and their stoichiometry within isolated complexes

• Co-immunoprecipitation: Using antibodies against ATP9 or other subunits to pull down interaction partners

These techniques can reveal how ATP9 participates in c-ring formation and interfaces with other components of the F₀ domain.

How does ATP9 contribute to mitochondrial membrane architecture in Y. lipolytica?

ATP9 involvement in mitochondrial membrane architecture involves:

• Dimer formation: ATP9 participates in ATP synthase dimerization, which induces membrane curvature essential for cristae formation

• Membrane bending: The specific arrangement of ATP9 within dimeric ATP synthase complexes creates localized membrane curvature

• Structural organization: Complete ATP synthase dimers containing ATP9 have been shown to reveal "the previously unknown subunit architecture of the dimer interface in the membrane, thereby providing major new insights into mitochondrial membrane architecture"

Research utilizing cryo-electron microscopy of purified ATP synthase complexes from Y. lipolytica has been instrumental in understanding these structural relationships.

What genetic engineering approaches can modify Y. lipolytica ATP9 expression?

For genetic manipulation of ATP9 expression in Y. lipolytica:

• Multi-copy integration: Y. lipolytica can be engineered to incorporate multiple expression cassettes through integrative transformation methods

• Promoter selection: The isocitrate lyase promoter (pICL1) has proven effective for heterologous protein expression in Y. lipolytica

• Integration targeting: rDNA or LTR zeta sequences of Ylt1 can be used as integration targets

• Selection strategies: The ura3d4 marker enables selection of multi-copy transformants

• Diploidisation: Combining different expression cassettes through diploidisation of selected haploid multi-copy transformants

These approaches have successfully generated Y. lipolytica strains expressing multiple heterologous proteins simultaneously, a technique potentially applicable to ATP9 expression manipulation.

How can ATP9 be utilized in studying mitochondrial genetic code variations?

The Y. lipolytica mitochondrial genome uses the standard mould mitochondrial genetic code, with an interesting exception related to CGN (arginine) codons:

• Codon usage analysis: No tRNAs capable of reading CGN codons exist in the Y. lipolytica mitochondrial genome

• Comparative genomics: CGN codons are absent in exonic open reading frames but present in intronic open reading frames

• Evolutionary implications: Several intronic open reading frames containing CGN codons have accumulated mutations and become pseudogenes, potentially due to these untranslatable codons

This makes Y. lipolytica ATP9 and other mitochondrial genes valuable for studying evolutionary mechanisms of genetic code adaptations and constraints.

What are common challenges in recombinant ATP9 expression and how can they be addressed?

Membrane protein expression challenges with ATP9 include:

These approaches have been successfully applied to other mitochondrial membrane proteins and can be adapted for ATP9 expression .

What analytical techniques are most informative for ATP9 functional characterization?

For comprehensive functional characterization of ATP9:

• Reconstitution into liposomes: Allows assessment of proton translocation function

• ATP hydrolysis/synthesis assays: Measures activity when incorporated into complete ATP synthase complexes

• Circular dichroism spectroscopy: Determines secondary structure elements and confirms proper folding

• Thermal shift assays: Evaluates protein stability under various buffer conditions

• Site-directed mutagenesis: Identifies critical residues for c-ring formation and proton translocation

These complementary approaches provide a multi-faceted assessment of ATP9 structural integrity and functional capacity.