Recombinant Lactuca sativa ATP synthase subunit b, chloroplastic (atpF)

What is the structure and function of ATP synthase subunit b (atpF) in Lactuca sativa chloroplasts?

ATP synthase subunit b, encoded by the atpF gene, is a critical component of the chloroplastic ATP synthase complex in lettuce. This protein is part of the membrane-embedded Fo portion of ATP synthase that forms a proton channel across the thylakoid membrane. The function of this subunit is mechanically coupled to the rotation of the c-ring, which drives ATP synthesis in the F1 region where catalysis of ADP + Pi → ATP occurs at the three α-β subunit interfaces .

For structural characterization, researchers typically employ techniques such as:

• Circular dichroism spectroscopy to confirm alpha-helical secondary structure

• Sequence alignment with homologous proteins from well-characterized species

• Computational modeling based on crystal structures from other organisms

• Membrane protein topology analysis to identify transmembrane domains

ATP synthase produces the adenosine triphosphate required for photosynthetic metabolism, with the synthesis mechanically coupled to proton translocation across the thylakoid membrane along an electrochemical gradient .

How does the atpF gene organization in Lactuca sativa compare to other plant species?

The atpF gene in many plants contains an intron, though this feature varies across species. Based on comparative genomics:

• In some plant lineages, including certain members of Malpighiales, the atpF intron has been lost

• The atpF gene is part of a conserved ATP synthase transcriptional unit consisting of atpI/H/F/A genes

• RNA editing sites may be present, particularly at codon 31 where C-U editing often occurs

To experimentally determine atpF gene structure in lettuce, researchers should:

• Design PCR primers flanking potential intron sites (e.g., atpF-1F and atpF-ISP-R as used in other species)

• Amplify the region using genomic DNA as template

• Sequence the products to confirm intron presence/absence and identify potential RNA editing sites

• Compare results with closely related Asteraceae family members

What expression systems are most suitable for producing recombinant Lactuca sativa atpF protein?

Based on successful approaches with other plant ATP synthase components, several expression systems can be considered:

When expressing Lactuca sativa atpF in E. coli:

• Optimize codon usage for bacterial expression

• Consider lower induction temperatures (16-20°C) to improve folding

• Test various induction conditions (IPTG concentration, duration)

• Include solubility enhancers in the buffer

The recombinant approach enables production of significant quantities of highly purified protein for structural and functional studies . The expression conditions can be verified through immunoblotting with antibodies specific to atpF or using fusion-tag detection methods .

What purification strategies yield the highest purity and functional integrity of recombinant Lactuca sativa atpF protein?

Purification of recombinant atpF protein typically involves multiple chromatographic steps:

• Initial preparation:

  • Cell lysis with buffer containing protease inhibitors (e.g., 20 mM Tris-HCl pH 8.0, 2% v/v Protease Inhibitor Cocktail)

  • Treatment with lysozyme (1 mg/mL) followed by sonication

• Chromatographic separation:

  • Affinity chromatography using fusion partner (MBP, His-tag, FLAG)

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography as a final polishing step

• Quality assessment:

  • SDS-PAGE analysis (12% polyacrylamide gel) to verify purity

  • Western blotting to confirm identity

  • Circular dichroism to confirm proper secondary structure

For membrane proteins like atpF, detergent selection is critical for maintaining structural integrity during purification. Effectiveness of the purification can be monitored through gel electrophoresis and immunoblotting methods, with comparison to native proteins as positive controls .

How can RNA editing sites in Lactuca sativa atpF be experimentally identified and verified?

RNA editing in atpF transcripts involves C-to-U conversions that can alter the encoded amino acids. To identify these sites:

• Experimental approach:

  • Extract total RNA from Lactuca sativa chloroplasts

  • Synthesize cDNA using reverse transcription

  • Amplify atpF cDNA using specific primers

  • Compare genomic DNA and cDNA sequences to identify differences

  • Focus particularly on codon 31, where C-U editing commonly occurs to convert proline to leucine

• Validation methods:

  • High-throughput sequencing of both DNA and RNA

  • Poison primer extension assays

  • REL (RNA Editing Site Loss) PCR

  • Northern blotting with editing site-specific probes

• Functional verification:

  • Express edited and unedited versions of the protein

  • Compare structural and functional properties

  • Assess impacts on interactions with other ATP synthase subunits

RNA editing is particularly important in chloroplast genes as it often restores conserved amino acids that are critical for protein function .

What techniques are most effective for reconstituting functional ATP synthase complexes using recombinant Lactuca sativa atpF?

Reconstitution of functional ATP synthase requires:

• Component preparation:

  • Purified recombinant atpF protein

  • Other ATP synthase subunits (either recombinant or isolated from native sources)

  • Appropriate lipids for liposome formation (typically phosphatidylcholine and phosphatidic acid)

• Reconstitution methods:

  • Detergent dialysis approach

  • Direct incorporation during liposome formation

  • Step-wise addition of components to optimize assembly

• Functional assessment:

  • ATP synthesis assays using artificial proton gradients

  • Proton pumping assays with pH-sensitive fluorescent dyes

  • Rotational analysis using single-molecule techniques

What strategies are most effective for site-directed mutagenesis of Lactuca sativa atpF to study structure-function relationships?

For site-directed mutagenesis of Lactuca sativa atpF, researchers can employ:

• Mutagenesis methodologies:

  • QuikChange site-directed mutagenesis

  • Gibson Assembly for larger modifications

  • Golden Gate cloning for multiple simultaneous mutations

  • CRISPR/Cas9 for plastid genome editing in vivo

• Strategic target selection:

  • Conserved residues identified through multi-species alignments

  • Residues in transmembrane domains implicated in proton translocation

  • Protein-protein interaction interfaces with other ATP synthase subunits

  • RNA editing sites (particularly codon 31) to create pre-edited versions

• Functional validation approaches:

  • Expression and purification to assess protein stability

  • Reconstitution assays to determine impact on ATP synthase activity

  • Structural analysis to evaluate conformational changes

Mutations can provide valuable insights into the mechanism of proton translocation and the structural features that determine c-ring stoichiometry .

How can chloroplast transformation be optimized for studying recombinant atpF in Lactuca sativa?

Chloroplast transformation offers advantages for studying atpF in its native context:

• Transformation methods for Lactuca sativa:

  • Biolistic particle delivery (gold particle bombardment)

  • PEG-mediated transformation of protoplasts

  • Selection using spectinomycin resistance genes

• Vector design considerations:

  • Homologous recombination regions flanking the atpF gene

  • Inclusion of the entire atpI/H/F/A transcriptional unit to maintain native regulation

  • Addition of epitope tags for detection and purification

  • Consideration of RNA editing sites in the construct design

• Validation approaches:

  • PCR verification of transgene integration

  • Southern blotting to confirm homoplasmy

  • RT-PCR and western blotting to assess expression

  • Phenotypic analysis of transformants for photosynthetic efficiency

Successful transformation of Lactuca sativa has been reported, providing a framework for chloroplast engineering in this species .

What is the relationship between atpF intron loss and RNA editing, and how can this inform genetic engineering approaches?

The relationship between intron presence and RNA editing in atpF has important implications:

• Evolutionary patterns observed:

  • In some plant lineages, atpF intron loss correlates with loss of RNA editing at codon 31

  • This suggests possible RNA-mediated gene conversion during evolution

  • The association between intron absence and RNA editing loss is consistent across the Malpighiales but not universal in other plant groups

• Design considerations for genetic constructs:

  • If Lactuca sativa atpF contains an intron, ensure proper splicing motifs are preserved

  • Consider pre-edited constructs to bypass RNA editing requirements

  • Test both intron-containing and intronless constructs for expression efficiency

• Experimental approaches to study this relationship:

  • Compare expression of constructs with and without introns

  • Assess RNA editing efficiency in different genetic backgrounds

  • Investigate whether splicing factors and editing factors interact

Understanding these processes enables more effective genetic engineering and can provide insights into chloroplast genome evolution mechanisms .

How does the c-ring stoichiometry of Lactuca sativa ATP synthase compare to other plant species, and what does this reveal about bioenergetic efficiency?

The c-ring stoichiometry (number of c-subunits forming the ring) directly affects the H+/ATP ratio and thus the bioenergetic efficiency:

• Determination methods:

  • Cryo-electron microscopy of isolated c-rings

  • Atomic force microscopy of reconstituted complexes

  • Native mass spectrometry of intact c-rings

  • Functional measurements of H+/ATP ratios

• Comparative analysis:

  • C-ring stoichiometry varies across species (typically 8-15 subunits)

  • Higher numbers of c-subunits result in more protons required per ATP

  • This represents an evolutionary adaptation to different environmental conditions

• Implications for Lactuca sativa:

  • The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits

  • This ratio directly impacts photosynthetic efficiency under different light conditions

  • Adaptation to agricultural conditions may have influenced optimal stoichiometry

Investigating these parameters enables understanding of how ATP synthase structure has evolved to optimize energy conversion in specific environmental niches.

What methods are most effective for assessing interactions between recombinant atpF and other ATP synthase subunits?

To characterize subunit interactions within the ATP synthase complex:

• In vitro binding assays:

  • Pull-down assays with tagged recombinant proteins

  • Surface plasmon resonance (SPR) for kinetic and affinity measurements

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Blue native PAGE for complex assembly analysis

• Structural approaches:

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to map binding regions

  • FRET analysis of labeled subunits

• Functional interaction studies:

  • Reconstitution with different combinations of subunits

  • Mutational analysis of putative interaction sites

  • Competition assays with peptide fragments

Understanding these interactions is crucial as ATP synthesis is mechanically coupled to the rotation of c-subunits, which drives the catalysis through the γ-rotation in the F1 region .

How do environmental factors affect expression and activity of atpF in Lactuca sativa, and how can this be studied with recombinant systems?

Environmental regulation of atpF expression and activity can be investigated through:

• Expression analysis approaches:

  • RT-qPCR under various conditions (light intensity, temperature, drought)

  • Proteomics to quantify protein levels

  • Reporter gene fusions to visualize expression patterns

  • Run-on transcription assays to assess transcriptional regulation

• Post-translational modification studies:

  • Phosphoproteomic analysis under different conditions

  • RNA editing efficiency assessment across environmental variables

  • Protein turnover studies using pulse-chase experiments

• Functional impact evaluation:

  • ATP synthesis rate measurements under varying conditions

  • Proton gradient formation analysis

  • Electron transport chain coupling efficiency

This research is particularly relevant for agricultural applications, as optimizing ATP synthase function could potentially enhance photosynthetic efficiency and crop productivity under variable environmental conditions.

What evolutionary insights can be gained from studying atpF sequence and structure across Asteraceae species compared to Lactuca sativa?

Comparative evolutionary analysis of atpF across Asteraceae can reveal:

• Sequence-based analyses:

  • Phylogenetic reconstruction based on atpF sequences

  • Selection pressure analysis (dN/dS ratios) to identify conserved functional domains

  • Identification of lineage-specific adaptations

  • Correlation of sequence changes with ecological niches

• Structural feature comparison:

  • Intron retention/loss patterns across Asteraceae

  • RNA editing site conservation or divergence

  • Protein secondary structure prediction comparisons

  • Transmembrane domain conservation

• Functional implications:

  • Correlation of c-ring stoichiometry with environmental adaptations

  • Analysis of species-specific post-translational modifications

  • Assessment of ATP synthase efficiency across related species

This research provides context for understanding the specific adaptations in Lactuca sativa ATP synthase that may relate to its agricultural performance.

How have RNA editing patterns in atpF evolved across plant lineages, and what does this reveal about Lactuca sativa atpF function?

RNA editing evolution provides insights into functional constraints:

• Comparative pattern analysis:

  • RNA editing site mapping across plant phylogeny

  • Correlation with intron presence/absence

  • Analysis of editing site gains and losses

  • Focus on the conserved C-U editing at codon 31 (P→L conversion)

• Mechanistic investigations:

  • Identification of cis-regulatory elements for editing machinery

  • Comparison of editing efficiency across species

  • Assessment of editing factors conservation

• Functional correlation:

  • Expression of edited and unedited versions to assess functional differences

  • Structural modeling to predict impacts of edited amino acids

  • Analysis of protein-protein interactions with both versions

The correlation between RNA editing loss and intron loss observed in some lineages suggests possible RNA-mediated gene conversion mechanisms in chloroplast genome evolution .

How has the atpF gene's inclusion in the atpI/H/F/A transcriptional unit influenced its evolution and expression in Lactuca sativa?

The organization of atpF within a polycistronic transcriptional unit has important implications:

• Transcriptional analysis approaches:

  • RT-PCR to identify processing of polycistronic transcripts

  • 5' and 3' RACE to map transcript termini

  • Northern blotting to quantify relative abundance of different transcript forms

  • Analysis of promoter elements controlling the operon

• Regulatory mechanisms:

  • Post-transcriptional processing patterns of the atpI/H/F/A transcript

  • Differential stability of processed transcripts

  • Coordination of expression with other ATP synthase components

  • Comparison of translation efficiency across the operon

• Evolutionary considerations:

  • Conservation of gene order across plant lineages

  • Co-evolution of genes within the transcriptional unit

  • Potential for horizontal gene transfer events

  • Impact of genome rearrangements on expression patterns

Understanding the coordination of expression within this transcriptional unit provides insights into the regulation of ATP synthase assembly and stoichiometry of components.