Recombinant Gibberella zeae Presequence translocated-associated motor subunit PAM17, mitochondrial (PAM17)

What is PAM17 and what is its significance in mitochondrial function?

PAM17 (Presequence translocated-associated motor subunit 17) is a critical component of the mitochondrial protein import machinery. It functions as the sixth motor subunit of the presequence translocase-associated motor (PAM) that drives the completion of preprotein translocation into the mitochondrial matrix. The significance of PAM17 lies in its role in regulating the organization of the Pam16-Pam18 complex, which in turn modulates the ATPase activity of matrix heat shock protein 70 (mtHsp70) at the inner membrane translocation site. Without proper PAM function, mitochondria would be incapable of importing essential matrix proteins required for mitochondrial processes like respiration, metabolism, and organelle maintenance . Research indicates that PAM17 anchors in the inner mitochondrial membrane and is exposed to the matrix, allowing it to facilitate protein import through strategic interactions with other PAM components.

How does PAM17 in Gibberella zeae compare structurally to PAM17 in other fungal species?

Gibberella zeae PAM17 (encoded by gene FGRRES_05029/FGSG_05029) consists of a mature protein spanning amino acid positions 62-235 with distinctive structural features . When comparing G. zeae PAM17 with other fungal homologs, researchers should focus on analyzing the conservation of key functional domains and transmembrane regions. The amino acid sequence (VSDKPQPETVQATPQPAPSNVLPPLDWNSFFKLRVKRRRYQMLFSITNGIFAGSGGAIFLS TGSAEPIISQIPLDPFMTLGLMTLAFSGLGWLSGPSVGNQVFYILNRQWKKQMTQKEAI FFERIKRNRVDPTNSSANNPVPDFYGEKISSVAGYRSWLKDQKAFNKKKTANFV) reveals transmembrane motifs and functional regions that can be analyzed via multiple sequence alignment techniques with other fungal species . Comparative structural analysis through techniques like homology modeling would reveal evolutionary conserved regions versus species-specific adaptations, potentially illuminating functional differences in protein import mechanisms across fungal lineages.

What are the optimal expression systems for producing recombinant G. zeae PAM17 protein?

The optimal expression system for recombinant G. zeae PAM17 production is the E. coli-based in vitro expression system, which has been validated for successfully generating full-length mature PAM17 protein . When designing expression constructs, researchers should consider:

• Inclusion of an N-terminal 10xHis tag to facilitate purification via affinity chromatography

• Expression of the mature protein region (amino acids 62-235) rather than the full-length protein to avoid potential issues with signal peptide processing

• Optimization of codon usage for E. coli to enhance expression levels

• Selection of appropriate vector backbones containing tightly regulated promoters

For optimal protein yield and stability, express the protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain protein integrity during storage and handling . Researchers should validate protein expression through Western blotting, mass spectrometry, and functional assays to confirm proper folding and activity of the recombinant protein.

What methodological approaches are most effective for studying PAM17 interactions with other mitochondrial import components?

To effectively study PAM17 interactions with other mitochondrial import components, researchers should employ a multi-faceted methodological approach:

• Co-immunoprecipitation (Co-IP): Using antibodies against tagged PAM17 to pull down interaction partners, particularly focusing on Pam16-Pam18 complex members and other PAM components

• Blue native polyacrylamide gel electrophoresis (BN-PAGE): For analyzing intact protein complexes and determining the molecular weight of the complex containing PAM17

• Yeast two-hybrid (Y2H) assays: For mapping the specific domains involved in protein-protein interactions

• In vitro binding assays: Using purified recombinant proteins to validate direct physical interactions and measure binding affinities

• Chemical crosslinking coupled with mass spectrometry: To capture transient interactions and identify precise interaction sites

Research has established that PAM17 is required for the formation of a stable complex between cochaperones Pam16 and Pam18, and promotes the association of Pam16-Pam18 with the presequence translocase . Study designs should include both wild-type and mutant forms of PAM17 to identify critical amino acid residues involved in these interactions, potentially through systematic alanine scanning mutagenesis.

How can researchers experimentally determine the specific role of PAM17 in mitochondrial protein import?

To experimentally determine PAM17's specific role in mitochondrial protein import, researchers should implement the following methodological approaches:

• Gene deletion/knockdown studies: Generate PAM17-deficient mutants to analyze the resulting phenotypes and mitochondrial protein import deficiencies. Mitochondria lacking PAM17 show selective impairment in matrix protein import and reduced import-driving activity of PAM .

• In vitro import assays: Use isolated mitochondria from wild-type and PAM17-deficient cells to compare import efficiency of radiolabeled precursor proteins. This quantitative approach allows researchers to determine:

  • Import kinetics (rate of protein translocation)

  • Substrate specificity (which proteins are most affected by PAM17 deficiency)

  • Energy requirements (ATP and membrane potential dependence)

• Complementation experiments: Reintroduce wild-type or mutant versions of PAM17 into deficient cells to identify functional domains required for activity.

• Real-time imaging: Utilize fluorescently tagged precursor proteins to visualize import processes in living cells with and without PAM17.

• Crosslinking studies: Apply chemical crosslinkers during active import to capture PAM17 interactions with translocating precursor proteins or other import machinery components.

These experimental approaches should be combined with structural analysis techniques to correlate functional observations with specific structural features of the PAM17 protein.

How does temperature stress affect PAM17 expression and function in Gibberella zeae?

Temperature stress significantly modulates PAM17 expression in Gibberella zeae, as revealed through transcriptomic analysis. RNA-seq data from the strain Z-3639 demonstrated differential expression patterns when comparing normal temperature conditions (25°C) versus heat stress conditions (37°C) . When designing experiments to investigate temperature effects on PAM17:

• Time-course expression analysis: Monitor PAM17 transcript and protein levels at different time points after temperature shift (15 minutes, 30 minutes, 1 hour, 2 hours) to capture both immediate and adaptive responses.

• Subcellular localization studies: Determine if temperature affects PAM17 localization within mitochondria using immunofluorescence or GFP-tagged constructs.

• Functional import assays: Compare mitochondrial protein import efficiency at different temperatures in wild-type versus PAM17-mutant strains.

• Protein-protein interaction analysis: Assess whether temperature affects PAM17's ability to facilitate formation of the Pam16-Pam18 complex using co-immunoprecipitation at different temperatures.

These approaches would provide insights into how G. zeae adapts its mitochondrial import machinery during temperature fluctuations, which may be particularly relevant given its pathogenicity toward crops across varying environmental conditions .

What are the differences between PAM17's roles in protein import compared to Tim44?

The functional relationship between PAM17 and Tim44 represents a sophisticated sequential action in mitochondrial protein import. Their distinct but complementary roles have been elucidated through genetic and biochemical analyses revealing synthetic interactions between PAM17 and TIM44 genes .

The sequential action of these proteins enables the coordinated translocation of preproteins across the inner mitochondrial membrane. PAM17 first facilitates the correct organization of the Pam16-Pam18 complex and promotes its association with the presequence translocase . Following this initial step, Tim44 positions mtHsp70 at the import channel, enabling the ATP-dependent binding of mtHsp70 to the incoming preprotein .

Sophisticated research approaches involving double mutant analyses have revealed that these proteins cooperate in a complementary manner, with PAM17 deficiency symptoms being exacerbated by Tim44 mutations . This indicates potential parallel pathways or compensatory mechanisms within the mitochondrial import machinery that warrant further investigation using techniques like chemical genetics or synthetic genetic array analysis.

How can researchers resolve contradictory data regarding PAM17 essentiality across different fungal species?

Resolving contradictory data regarding PAM17 essentiality across fungal species requires a systematic comparative analysis approach:

• Standardized knockout methodology: Implement CRISPR-Cas9 or homologous recombination techniques using identical targeting strategies across multiple fungal species to ensure comparable gene disruption efficiency.

• Growth condition matrix assessment: Evaluate PAM17 knockout strains under a comprehensive matrix of growth conditions including:

  • Temperature ranges (15°C, 25°C, 30°C, 37°C)

  • Carbon sources (glucose, glycerol, ethanol, acetate)

  • Nitrogen sources (amino acids, ammonium)

  • Osmotic stress conditions

  • Oxidative stress conditions

• Compensatory mechanism identification: Perform RNA-seq and proteomics analyses on PAM17-deficient strains across species to identify differentially regulated genes/proteins that might compensate for PAM17 loss.

• Phylogenetic analysis: Correlate PAM17 sequence divergence with functional essentiality across the fungal kingdom to identify potential evolutionary adaptation points.

• Domain swapping experiments: Create chimeric PAM17 proteins containing domains from species where it is essential versus non-essential to pinpoint regions responsible for functional differences.

A common source of contradictory results involves the varying ability of different species to activate alternative import pathways when PAM17 is absent. Implementing these systematic approaches would help distinguish between true biological variation in PAM17 essentiality versus methodological artifacts or incomplete phenotypic assessment.

How can researchers overcome challenges in purifying functional recombinant PAM17 protein?

Purifying functional recombinant PAM17 presents several challenges due to its membrane-associated nature. Researchers can implement the following optimization strategies:

• Expression optimization:

  • Use the validated E. coli expression system with N-terminal 10xHis-tag

  • Test multiple E. coli strains (BL21(DE3), C41(DE3), Rosetta) to identify optimal host

  • Vary induction conditions (temperature, IPTG concentration, induction time)

  • Consider codon optimization specifically for membrane protein expression

• Solubilization strategies:

  • Screen different detergents (n-Dodecyl β-D-maltoside, CHAPS, Triton X-100) at various concentrations

  • Test detergent-lipid mixtures for maintaining native-like environment

  • Consider mild solubilization conditions to preserve protein-protein interactions

• Purification refinement:

  • Implement two-step purification (IMAC followed by size exclusion chromatography)

  • Use additives in purification buffers (6% trehalose, glycerol, reducing agents)

  • Maintain pH 8.0 throughout purification process as indicated optimal

• Functional validation:

  • Develop in vitro assays to confirm proper folding and activity

  • Use circular dichroism to assess secondary structure integrity

  • Employ thermal shift assays to evaluate protein stability

• Storage optimization:

  • Store as lyophilized powder for extended shelf life (up to 12 months at -20°C/-80°C)

  • For liquid storage, use small aliquots to avoid freeze-thaw cycles

  • Include stabilizers like trehalose in storage buffers

By systematically addressing these aspects, researchers can significantly improve the yield and functionality of purified recombinant PAM17 protein for downstream applications.

What are the best approaches for analyzing PAM17 function in the context of plant pathogenicity of Gibberella zeae?

Analyzing PAM17 function in the context of G. zeae (Fusarium graminearum) pathogenicity requires specialized methodological approaches that bridge molecular biology with plant pathology:

• Gene deletion and complementation:

  • Generate PAM17 knockout mutants using CRISPR-Cas9 or homologous recombination

  • Create complemented strains with native promoter-driven expression

  • Develop conditional expression systems to study PAM17 function at different infection stages

• Infection model systems:

  • Use wheat head, barley spike, and maize ear infection assays to quantify virulence differences

  • Develop microscopy-based techniques to visualize fungal colonization patterns in plant tissues

  • Implement detached leaf assays for high-throughput preliminary screening

• Environmental stress correlation:

  • Analyze PAM17 expression under conditions mimicking plant infection (pH changes, oxidative stress, plant defense compounds)

  • Compare expression patterns between normal (25°C) and stress (37°C) conditions as observed in transcriptomic studies

  • Correlate heat stress responses with in planta expression patterns

• Mitochondrial function assessment:

  • Measure reactive oxygen species production in wild-type versus PAM17 mutants during plant infection

  • Assess mitochondrial protein import efficiency in fungal cells isolated from infected plant tissue

  • Evaluate mitochondrial morphology and distribution during host colonization

• Omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data to build comprehensive models of PAM17's role in pathogenicity

  • Use systems biology approaches to identify connections between mitochondrial function and virulence factors

These methodologies would help establish whether PAM17-dependent mitochondrial functions play significant roles in the pathogenicity mechanisms of G. zeae, potentially identifying new targets for disease control strategies.

How should researchers interpret changes in PAM17 expression during different developmental stages of Gibberella zeae?

Interpreting changes in PAM17 expression across G. zeae developmental stages requires a structured analytical framework:

• Normalization and reference gene selection:

  • Use multiple reference genes validated for stability across developmental stages

  • Apply geometric averaging of reference genes for optimal normalization

  • Consider developmental stage-specific reference genes when appropriate

• Statistical analysis approaches:

  • Implement ANOVA with post-hoc tests for multi-stage comparisons

  • Use time-series analysis methods for continuous developmental monitoring

  • Apply non-parametric tests for data not meeting normality assumptions

• Data visualization techniques:

  • Generate heat maps correlating PAM17 expression with other mitochondrial import components

  • Use principal component analysis to identify developmental stage clusters

  • Create expression profile plots across key developmental transitions

• Biological interpretation framework:

  • Correlate expression changes with known metabolic shifts during development

  • Map expression patterns to morphological transitions (mycelial growth, sporulation, germination)

  • Consider transcription factor binding site analysis to identify potential regulators

• Validation approaches:

  • Confirm transcript-level changes with protein-level measurements

  • Use reporter gene constructs to visualize expression in developing structures

  • Perform targeted perturbation at specific developmental stages to confirm functional significance

The interpretation should consider that PAM17 expression might correlate with changing energy demands during development, particularly during transitions requiring extensive mitochondrial biogenesis or remodeling. Changes observed during temperature stress (37°C vs 25°C) may provide insights into how developmental regulation intersects with environmental adaptation.

What bioinformatic approaches are most effective for identifying potential PAM17 interaction partners?

Identifying potential PAM17 interaction partners requires integrating multiple bioinformatic approaches:

• Sequence-based methods:

  • Analyze conserved interaction motifs within the PAM17 amino acid sequence (VSDKPQPETVQATPQPAPSNVLPPLDWNSFFKLRVKRRRYQMLFSITNGIFAGSGGAIFL STGSAEPIISQIPLDPFMTLGLMTLAFSGLGWLSGPSVGNQVFYILNRQWKKQMTQKEAI FFERIKRNRVDPTNSSANNPVPDFYGEKISSVAGYRSWLKDQKAFNKKKTANFV)

  • Implement coevolution analysis to identify proteins that show synchronized evolutionary patterns

  • Use domain-based interactome prediction tools focused on mitochondrial proteins

• Structural modeling approaches:

  • Generate homology models based on known structures of PAM components

  • Perform protein-protein docking simulations with candidate partners

  • Use molecular dynamics simulations to evaluate stability of predicted interactions

• Network-based methods:

  • Apply guilt-by-association algorithms using existing interaction databases

  • Construct co-expression networks from transcriptomic data, particularly under stress conditions

  • Implement Bayesian integration of multiple data types for confidence scoring

• Comparative genomics strategies:

  • Identify synteny patterns of PAM17 and potential partners across fungal species

  • Use phylogenetic profiling to find proteins with similar evolutionary presence/absence patterns

  • Analyze shared regulatory elements suggesting coordinated expression

• Data visualization and prioritization:

  • Generate interaction network visualizations with confidence scoring

  • Implement clustering algorithms to identify functional modules

  • Prioritize candidates based on multiple evidence types

Known interactions between PAM17 and the Pam16-Pam18 complex and its relationship with Tim44 provide anchor points for expanding the interaction network. Researchers should focus on identifying both stable structural interactions and transient functional interactions that may occur only during active protein import.

What are the most promising research avenues for understanding PAM17's role in fungal adaptation to environmental stresses?

The most promising research avenues for understanding PAM17's role in fungal environmental adaptation include:

• Comparative transcriptomics across stress conditions:

  • Expand beyond heat stress (37°C) to include cold, osmotic, oxidative, and pH stresses

  • Compare stress responses between saprophytic and plant infection phases

  • Analyze PAM17 expression patterns in natural field conditions versus laboratory settings

• Stress-specific protein interaction mapping:

  • Implement proximity labeling techniques under different stress conditions

  • Identify stress-specific changes in the PAM17 interactome

  • Characterize how interactions with Pam16-Pam18 complex are modified by stress

• Mitochondrial dynamics and stress adaptation:

  • Investigate relationships between PAM17 function and mitochondrial fission/fusion under stress

  • Analyze mitochondrial proteome remodeling in response to stress in wild-type versus PAM17 mutants

  • Assess mitochondrial morphology changes and their dependence on functional PAM17

• Cross-species functional conservation:

  • Compare PAM17 function across diverse fungal species with different ecological niches

  • Determine if PAM17's role in stress adaptation is conserved from saprophytic to pathogenic fungi

  • Perform complementation studies with PAM17 from different species to identify adaptations

• Integration with cellular stress response pathways:

  • Map connections between PAM17 and known stress signaling pathways (MAPK, TOR, HOG)

  • Identify transcription factors regulating PAM17 expression under stress

  • Investigate post-translational modifications of PAM17 during stress responses

RNA-seq analysis of F. graminearum under heat stress has already provided valuable insights into transcriptional responses to temperature changes . Building on this foundation, researchers can develop a comprehensive understanding of how mitochondrial protein import adaptation via PAM17 contributes to the remarkable environmental adaptability of fungi like G. zeae.

How can structural biology approaches advance our understanding of PAM17 function?

Structural biology approaches offer significant potential to advance PAM17 functional understanding:

Recent advances in cryo-EM have revolutionized the structural biology of membrane protein complexes, making this an especially promising approach for understanding PAM17's integration into the larger mitochondrial import machinery. Structural studies would provide mechanistic insights into how PAM17 organizes the Pam16-Pam18 complex and contributes to the regulation of mtHsp70 activity at the inner membrane translocation site .