Recombinant Ashbya gossypii Lon protease homolog, mitochondrial (PIM1), partial

What is the Lon protease homolog PIM1 in Ashbya gossypii?

PIM1 in A. gossypii is a mitochondrial ATP-dependent protease homologous to the well-characterized Lon protease family found across various organisms. It exhibits over 30% identity with ATP-dependent protease La from Escherichia coli, Lon from Bacillus brevis, and related proteases from other organisms . The protein functions within the mitochondrial matrix and plays crucial roles in mitochondrial genome maintenance and protein quality control.

How does A. gossypii PIM1 compare to S. cerevisiae PIM1?

A. gossypii PIM1 shares significant functional and structural similarities with S. cerevisiae PIM1. Both are mitochondrial ATP-dependent proteases involved in mitochondrial genome integrity. In S. cerevisiae, PIM1 is 1133 amino acids long with a putative mitochondrial import signal in the N-terminal region . Studies have shown that in both organisms, the absence of functional PIM1 leads to an inability to utilize nonfermentable carbon sources and maintain functional mitochondrial DNA . The extensive synteny between A. gossypii and S. cerevisiae genomes supports their functional conservation, making findings in one organism often applicable to the other .

What are the known roles of PIM1 in mitochondrial function?

PIM1 serves multiple essential functions in mitochondria:

• Maintenance of mitochondrial genome integrity

• Selective degradation of misfolded or damaged proteins in the mitochondrial matrix

• Cooperation with the mitochondrial Hsp70 system to prevent protein aggregation

• Regulation of mitochondrial gene expression by controlling the translation of genes like cytochrome c oxidase subunit I (CoxI) and cytochrome b (Cob)

• Involvement in the splicing of COXI and COB transcripts, particularly introns encoding mRNA maturases

• Response to thermal stress, suggesting a role in the heat shock response

Why is A. gossypii an important model organism for studying mitochondrial proteins?

A. gossypii is an increasingly important model organism for several reasons:

• It exhibits extensive synteny with the S. cerevisiae genome, facilitating comparative genomics

• It has a filamentous growth pattern useful for studying the evolution of fungal morphology

• It naturally overproduces riboflavin, making it industrially relevant

• Multiple genetic manipulation tools have been developed for A. gossypii, including CRISPR/Cas9 and CRISPR-Cpf1 systems

• Its genome has been completely sequenced and well-annotated, with telomere-to-telomere coverage of all 14 chromosome ends

What expression systems are most effective for producing recombinant A. gossypii PIM1?

Based on available data, recombinant A. gossypii PIM1 can be effectively expressed in several host systems:

Most commercially available recombinant PIM1 preparations use one of these systems, with purities typically greater or equal to 85% as determined by SDS-PAGE .

What are the critical considerations when designing expression constructs for A. gossypii PIM1?

When designing expression constructs for A. gossypii PIM1, researchers should consider:

• Inclusion of the appropriate mitochondrial targeting sequence if studying import mechanisms

• Selection of purification tags that won't interfere with PIM1's ATPase or protease activities

• Codon optimization based on the expression host

• Selection of a vector with an appropriate inducible promoter system

• Consideration of expressing full-length protein versus functional domains

For functional studies, it's crucial to determine whether to include or exclude the mitochondrial targeting sequence, as this affects cellular localization. When studying enzyme kinetics, the N-terminal protease domain and the C-terminal ATPase domain must both be properly folded for activity .

What purification methods yield the highest activity for recombinant PIM1?

Optimal purification strategies for maintaining PIM1 activity include:

• Affinity chromatography using nickel or cobalt resins for His-tagged PIM1

• Ion exchange chromatography as a secondary purification step

• Size exclusion chromatography for final polishing and buffer exchange

• Maintaining buffer conditions that preserve ATP binding capacity (typically including Mg²⁺)

• Including ATP or non-hydrolyzable ATP analogs during purification to stabilize the protein

• Avoiding harsh elution conditions that might denature the protein

Maintaining a temperature of 4°C throughout purification and including protease inhibitors in early purification steps is critical for preserving activity. Commercial preparations typically achieve ≥85% purity as determined by SDS-PAGE .

How can the ATPase activity of recombinant PIM1 be measured?

The ATPase activity of recombinant PIM1 can be measured using several methods:

• Malachite green phosphate assay: Measures inorganic phosphate released during ATP hydrolysis

  • Advantages: High sensitivity, suitable for kinetic measurements

  • Protocol: Incubate PIM1 with ATP, stop reaction with malachite green reagent, measure absorbance at 620-640 nm

• Coupled enzyme assay: Using pyruvate kinase and lactate dehydrogenase

  • Advantages: Continuous real-time monitoring of activity

  • Protocol: Measure decrease in NADH absorbance at 340 nm as ATP is regenerated from ADP

• Radioactive [γ-³²P]ATP assay: Measures release of radioactive phosphate

  • Advantages: Extremely sensitive, can detect very low activity levels

  • Protocol: Separate released phosphate by thin-layer chromatography and quantify by scintillation counting

When designing these experiments, include appropriate controls such as PIM1 without substrate, heat-inactivated PIM1, and known ATPase inhibitors to validate specificity .

What are reliable methods to assess the protease activity of recombinant PIM1?

Protease activity of recombinant PIM1 can be assessed through:

• Fluorogenic peptide substrates: Use commercially available Lon protease substrates with fluorescence resonance energy transfer (FRET) pairs

  • Advantages: Real-time kinetic measurements, high sensitivity

  • Protocol: Measure increase in fluorescence as the substrate is cleaved

• SDS-PAGE degradation assay: Monitor degradation of known substrate proteins

  • Advantages: Demonstrates activity against physiologically relevant substrates

  • Protocol: Incubate PIM1 with substrate protein in the presence of ATP, analyze by SDS-PAGE over time

• Western blot analysis: For detection of specific substrate degradation

  • Advantages: Highly specific, can monitor endogenous substrate levels

  • Protocol: Perform western blot analysis using antibodies against known PIM1 substrates

Critical controls should include reactions without ATP (as PIM1 is ATP-dependent), heat-inactivated PIM1, and specific protease inhibitors .

How can the role of PIM1 in mitochondrial quality control be studied experimentally?

The role of PIM1 in mitochondrial quality control can be studied through:

• Gene knockout/knockdown approaches:

  • Generate PIM1-deficient strains using CRISPR-Cpf1 system adapted for A. gossypii

  • Assess mitochondrial morphology and function in wild-type versus mutant strains

• Fluorescence microscopy with mitochondrial markers:

  • Label mitochondria with GFP using plasmids like pAgrMO-G1 (expressing GFP fused to S. cerevisiae Cox4)

  • Compare mitochondrial morphology and distribution in wild-type and PIM1-deficient cells

• Mitochondrial respiration analysis:

  • Measure oxygen consumption using respirometry

  • Compare ADP-stimulated (state 3) respiration in wild-type versus PIM1-deficient mitochondria

• Assessment of mitochondrial genome stability:

  • Analyze mtDNA integrity and maintenance in PIM1 mutants

  • Examine the ability of cells to grow on non-fermentable carbon sources

• Substrate identification studies:

  • Use proteomics approaches to identify proteins that accumulate in PIM1-deficient mitochondria

  • Perform co-immunoprecipitation to identify PIM1-interacting proteins

How can recombinant PIM1 be used to study mitochondrial protein import and processing?

Recombinant PIM1 can be instrumental in studying mitochondrial protein import through:

• In organello import assays:

  • Isolate mitochondria from wild-type and PIM1-deficient cells

  • Add radiolabeled precursor proteins and monitor their import and processing

  • Compare processing patterns to identify PIM1-dependent steps

• Reconstitution experiments:

  • Add recombinant PIM1 to isolated PIM1-deficient mitochondria

  • Test whether this restores normal processing of precursor proteins

  • Use site-directed mutants of PIM1 to identify critical residues for function

• Direct cleavage assays:

  • Incubate recombinant PIM1 with putative substrate proteins

  • Analyze cleavage products by mass spectrometry to identify precise cleavage sites

  • Compare with processing observed in intact mitochondria

These approaches have revealed that PIM1-mediated cleavage is coupled to import, such that reducing PIM1 activity can induce accumulation of proteins at the mitochondrial surface .

What insights can be gained from studying PIM1 in relation to mitochondrial disease models?

Studying PIM1 in relation to mitochondrial diseases can provide several key insights:

• Understanding basic mechanisms of mitochondrial protein quality control

• Identification of how defects in protein processing contribute to mitochondrial dysfunction

• Development of potential therapeutic strategies targeting protein quality control

Research has shown that PIM1/Lon protease is involved in the PINK1-Parkin pathway, which is implicated in Parkinson's disease. In human cells, the MPP (mitochondrial processing peptidase) is involved in PINK1 import and processing, affecting mitochondrial quality control. When mitochondria are damaged (depolarized), PINK1 accumulates on the mitochondrial surface and recruits Parkin, leading to mitophagy. Similar mechanisms may operate in fungal systems with PIM1 playing comparable roles .

How might PIM1 function relate to A. gossypii's natural overproduction of riboflavin?

A. gossypii is known for naturally overproducing riboflavin (vitamin B2), a property that has been exploited commercially. The relationship between PIM1 and riboflavin production may involve several mechanisms:

• Mitochondrial integrity and metabolism:

  • PIM1 is essential for maintaining mitochondrial genome integrity

  • Riboflavin production is linked to central metabolism and redox balance, which are influenced by mitochondrial function

  • In riboflavin-overproducing mutants, changes in oxidative stress response and aging of cells have been observed

• Protein quality control:

  • PIM1 regulates the stability of key metabolic enzymes

  • Some flavoproteins are located in mitochondria and may be regulated by PIM1

  • A homozygous mutation in AgILV2 gene (encoding acetohydroxyacid synthase, a flavoprotein in mitochondria) was found in a riboflavin-overproducing mutant

• Stress response:

  • Riboflavin overproduction in A. gossypii may be associated with aging of cells and response to oxidative stress

  • PIM1 expression increases after thermal stress, suggesting a role in stress response

These connections suggest that PIM1 function may indirectly influence riboflavin biosynthesis through its effects on mitochondrial function and stress response pathways.

What are common pitfalls when working with recombinant A. gossypii PIM1 and how can they be addressed?

Common challenges and solutions when working with recombinant PIM1 include:

Additionally, when studying mitochondrial functions with isolated organelles, ensure mitochondrial integrity is preserved by monitoring membrane potential and respiration capacity as control measures .

How should experimental conditions be optimized when studying PIM1's role in mitochondrial RNA processing?

When investigating PIM1's role in mitochondrial RNA processing, consider these optimization strategies:

• RNA extraction and preservation:

  • Use specialized extraction methods for mitochondrial RNA

  • Include RNase inhibitors to prevent degradation

  • Process samples quickly and maintain cold temperatures

• Comparison of wild-type and PIM1-deficient strains:

  • Generate clean knockout strains using CRISPR-Cpf1 system

  • Use conditional expression systems to study essential functions

  • Include complementation controls to confirm phenotype specificity

• Transcript analysis methods:

  • Use Northern blotting for specific transcripts

  • Apply RT-qPCR for quantitative analysis

  • Employ RNA-seq for genome-wide effects

• Splicing analysis:

  • Design primers spanning intron-exon junctions

  • Use PCR to amplify spliced and unspliced forms

  • Sequence products to confirm precise splicing defects

Research has shown that PIM1 mutants exhibit deficiencies in the splicing of COXI and COB transcripts, particularly introns encoding mRNA maturases, and these transcripts are degraded in the absence of PIM1 .

What controls are essential when studying the effects of PIM1 knockdown or knockout?

Critical controls for PIM1 knockdown/knockout studies include:

• Verification of knockdown/knockout efficiency:

  • Confirm at both mRNA level (RT-qPCR) and protein level (Western blot)

  • Quantify the degree of reduction in gene expression

• Phenotypic specificity controls:

  • Complementation with wild-type PIM1 to rescue phenotypes

  • Use of catalytically inactive PIM1 mutants as negative controls

  • Comparison with knockdowns of related mitochondrial proteases

• Assessment of mitochondrial integrity:

  • Monitor membrane potential using fluorescent dyes

  • Assess respiration capacity

  • Check levels and processing of mitochondrial proteins

  • Examine mitochondrial morphology by microscopy

• Functional controls:

  • Test growth on fermentable versus non-fermentable carbon sources

  • Assess mitochondrial genome stability

  • Measure ATP production capacity

How might multi-omics approaches be applied to elucidate the complete regulatory network of PIM1 in A. gossypii?

Advanced multi-omics strategies to investigate PIM1's regulatory network could include:

• Integrated genomics, transcriptomics, and proteomics:

  • Genome sequencing of wild-type and PIM1 mutant strains to identify genetic variants

  • RNA-seq to detect changes in gene expression and splicing patterns

  • Proteomics to identify changes in protein abundance and post-translational modifications

  • Integration of these datasets to construct comprehensive regulatory networks

• Metabolomics profiling:

  • Analysis of metabolite changes in PIM1 mutants

  • Focus on mitochondrial metabolites and riboflavin precursors

  • Correlation of metabolite levels with transcriptomic and proteomic changes

• Protein-protein interaction studies:

  • Immunoprecipitation coupled with mass spectrometry to identify interacting partners

  • Yeast two-hybrid screens or BioID proximity labeling

  • Validation of interactions through co-immunoprecipitation and functional assays

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Identify transcription factors affected by PIM1 activity

  • Map changes in chromatin accessibility and histone modifications

Studies on riboflavin-overproducing A. gossypii mutants have already employed some of these approaches, revealing mutations in genes involved in amino acid metabolism, the TCA cycle, and purine/pyrimidine metabolism that contribute to the phenotype .

What mechanisms might explain the differential sensitivity of various substrates to PIM1 protease activity?

Several sophisticated mechanisms may explain substrate selectivity of PIM1 protease:

• Structural recognition elements:

  • Specific amino acid sequences or structural motifs may serve as recognition sites

  • Conformational changes in substrates may expose or mask these recognition elements

  • Post-translational modifications might alter substrate recognition

• Co-chaperone interactions:

  • Interaction with mitochondrial Hsp70 systems may facilitate substrate delivery

  • Different co-chaperones may target specific substrate classes to PIM1

  • Competition between chaperones and PIM1 for binding to misfolded proteins

• Compartmentalization within mitochondria:

  • Microdomains within the mitochondrial matrix may concentrate PIM1 and specific substrates

  • Substrate localization may determine accessibility to PIM1

  • Membrane association may protect some proteins from degradation

• Allosteric regulation:

  • Binding of specific metabolites or signaling molecules may alter PIM1 conformation and substrate specificity

  • ATP levels may differentially affect recognition and processing of various substrates

  • Oligomeric state changes might influence substrate selection

Research has shown that PIM1-mediated proteolysis is remarkably sensitive to certain substrates, with even modest reductions in PIM1 levels causing significant accumulation of specific proteins while minimally affecting others .

How might comparative analysis of PIM1 function across evolutionary distant fungi provide insights into mitochondrial quality control mechanisms?

Comparative analysis of PIM1 across fungal species offers powerful insights:

• Evolutionary conservation and divergence:

  • Identification of conserved domains and residues critical for function

  • Discovery of species-specific adaptations in substrate recognition

  • Understanding how PIM1 function has evolved with changes in mitochondrial genome size and complexity

• Correlation with ecological niches:

  • Analysis of how PIM1 function differs between species with different lifestyles

  • Examination of adaptations in thermotolerant versus mesophilic fungi

  • Investigation of changes in oxidative stress response mechanisms

• Differential regulation:

  • Comparison of PIM1 expression regulation across species

  • Analysis of how stress responses involving PIM1 have evolved

  • Investigation of species-specific regulatory networks

• Functional complementation studies:

  • Cross-species complementation experiments to test functional conservation

  • Domain-swapping between orthologues to identify regions responsible for species-specific functions

  • Engineering of chimeric proteins to understand structure-function relationships

The research community has already begun such comparative work, with PIM1 homologs characterized in diverse fungi including S. cerevisiae, A. gossypii, and various other species, revealing both conserved functions in mitochondrial genome maintenance and species-specific adaptations .

What role might PIM1 play in coordinating the response to mitochondrial stress with nuclear gene expression?

The coordination between mitochondrial stress and nuclear gene expression involving PIM1 may occur through:

• Retrograde signaling pathways:

  • PIM1 dysfunction may generate specific signaling molecules

  • Accumulation of unprocessed proteins in PIM1-deficient mitochondria may trigger stress responses

  • These signals could modulate nuclear gene expression through dedicated pathways

• Dual-localization proteins:

  • Some proteins may shuttle between mitochondria and nucleus

  • PIM1 may regulate the abundance or processing of these dual-localized proteins

  • Changes in their localization or abundance could affect nuclear gene expression

• Metabolic intermediates as signals:

  • Mitochondrial dysfunction due to PIM1 deficiency may alter metabolite levels

  • These metabolites may serve as signals affecting nuclear transcription factors

  • Riboflavin or its precursors might serve as such signaling molecules in A. gossypii

• Integration with cell cycle regulation:

  • PIM1-dependent mitochondrial quality control may be coordinated with cell cycle progression

  • Defects in mitochondrial function could trigger cell cycle checkpoints

  • This coordination ensures proper mitochondrial inheritance during cell division

Studies in riboflavin-overproducing A. gossypii mutants have identified changes in gene expression and mutations in pathways that might be involved in such regulatory networks, suggesting complex interactions between mitochondrial function and nuclear gene expression .