Recombinant Scheffersomyces stipitis Vacuolar ATPase assembly integral membrane protein VMA21 (VMA21)

Basic Research Questions

• What is the function of VMA21 in Scheffersomyces stipitis and how does it compare to its homologs in other species?

VMA21 in Scheffersomyces stipitis functions as an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal proton pump complex. It is responsible for the assembly of the V-ATPase and its translocation to vacuoles/lysosomes .

The function appears conserved across species from yeast to humans. In humans, VMA21 is the diverged ortholog of yeast Vma21p, and like Vma21p, serves as an essential assembly chaperone of the V-ATPase . Mutations in human VMA21 cause X-linked myopathy with excessive autophagy (XMEA) , while in S. stipitis, it plays a critical role in maintaining proper vacuolar acidification which affects various cellular processes.

Methodologically, researchers can use comparative genomic approaches and sequence alignment tools to analyze conservation across species. The protein sequence for S. stipitis VMA21 (UniProt ID: A3LU53) can be aligned with homologs from other organisms to identify conserved domains.

• What are the structural characteristics of the VMA21 protein in S. stipitis?

The VMA21 protein in Scheffersomyces stipitis is a small integral membrane protein of 80 amino acids with the sequence: "MAAEIPTSVIQKLVFFTGAMIIFPIFTFFVCQYLFSNNALISGGIAALMANVVLIGYVVVAFTEDTSSLADEKVETKKDI" .

Structural analysis suggests:

• It contains multiple hydrophobic regions consistent with its role as an integral membrane protein

• It likely spans the membrane multiple times

• The full-length protein corresponds to the amino acid residues 1-80

To study its structure, researchers should consider membrane protein isolation techniques, followed by circular dichroism spectroscopy or crystallization attempts for X-ray crystallography. Due to the challenges with membrane protein crystallization, computational modeling approaches using homology modeling against known V-ATPase assembly factors could provide preliminary structural insights.

• What expression systems are most effective for recombinant production of S. stipitis VMA21?

For recombinant production of S. stipitis VMA21, researchers can utilize:

• Mammalian cell expression systems: These provide proper folding and post-translational modifications for membrane proteins, as demonstrated in commercial productions of recombinant VMA21 proteins .

• Yeast expression systems: Using S. cerevisiae as a host might be advantageous due to the conserved function of V-ATPase assembly factors across yeast species .

• E. coli systems with specialized tags: For small membrane proteins like VMA21, E. coli systems with fusion tags that enhance solubility can be employed, though refolding may be necessary.

Methodological considerations include using a His-tag for purification , employing Tris-based buffers with 50% glycerol for stability , and storing at -20°C to -80°C for long-term preservation.

Advanced Research Questions

• How do mutations in VMA21 affect V-ATPase function and what are the downstream metabolic consequences?

Mutations in VMA21 lead to V-ATPase misassembly and dysfunction, with several downstream consequences:

a) Lysosomal acidification impairment:

• Raises lysosomal pH, reducing lysosomal degradative ability

• Blocks autophagy, causing accumulation of undigested materials

• Results in reduced LysoTracker staining and decreased Lamp1 expression

b) Metabolic disruptions:

• Impaired lipophagy leads to lipid droplet accumulation in autolysosomes

• Triggers endoplasmic reticulum (ER) stress

• Causes sequestration of unesterified cholesterol in lysosomes

• Activates sterol response element-binding protein-mediated cholesterol synthesis pathways

c) Tissue-specific effects:

• In liver: hepatic steatosis, mild cholestasis, chronic elevation of aminotransferases, and elevated LDL cholesterol

• In muscle: formation of autophagic vacuoles with accumulated extracellular matrix components

Methodologically, researchers can use zebrafish models with CRISPR-Cas9 edited vma21 to study these effects, as they phenocopy human disease features including impaired motor function, liver dysfunction, and dysregulated autophagy .

• What techniques can be employed to assess the impact of VMA21 deficiency on lysosomal pH and autophagy?

Several experimental approaches can be used to assess VMA21 deficiency effects:

a) Lysosomal acidification assessment:

• LysoTracker Red staining to selectively mark acidic organelles

• Measurement of lysosomal pH using ratiometric fluorescent probes

b) Autophagy flux evaluation:

• Western blot analysis of LC3I and LC3II expression and determination of LC3II/LC3I ratio

• Fluorescent constructs like pTol2 (Ubbi:GFP-LC3-RFP-LC3ΔG) to evaluate autophagic flux through GFP:RFP ratios

• Immunofluorescence staining for lysosome-associated membrane protein-1 (Lamp1)

c) Ultrastructural analysis:

• Electron microscopy to identify autophagic vacuoles with electron-dense material and naked membranes

• Analysis of autolysosomes for lipid droplet accumulation

d) Functional assays:

• For liver dysfunction: BODIPY bile flux assays to assess cholestatic phenotype

• Touch-evoked escape response assays to evaluate motor function in zebrafish models

These methodologies can be applied to cell culture models with VMA21 knockdown or animal models with VMA21 mutations.

• How does VMA21 deficiency affect metabolic pathways in S. stipitis compared to other yeast species?

VMA21 deficiency impacts metabolic pathways differently across yeast species:

a) Comparative metabolic effects:

• S. stipitis is a Crabtree-negative yeast with fully respiratory metabolism under both glucose-limited and glucose-excess conditions

• In contrast, S. cerevisiae (Crabtree-positive) shows different metabolic patterns between respiratory and fermentative growth

b) Carbon metabolism differences:

• S. stipitis shows higher flux through the TCA cycle regardless of cultivation mode

• The flux through the oxidative part of the pentose phosphate pathway is higher in S. stipitis

• S. stipitis has greater respiratory capacity due to the presence of an alternative respiration system and Complex I

c) Metabolite accumulation patterns:

• S. stipitis shows significantly higher accumulation of certain metabolites like citramalate and phenylacetate compared to S. cerevisiae

• Metabolomic analysis reveals distinct profiles of glycolytic intermediates

Methodologically, researchers can use 13C-based flux analysis to determine the distribution of metabolic fluxes, combining with metabolomics approaches like capillary electrophoresis time-of-flight mass spectrometry to analyze intracellular metabolites .

• What are the potential therapeutic approaches for addressing VMA21 dysfunction based on current research?

Current research suggests several therapeutic approaches for VMA21 dysfunction:

a) Autophagy modulators:

• Edaravone and LY294002 (PI3K inhibitor) improved survival and motor function in zebrafish VMA21 mutants

• Multiple autophagy modulators showed beneficial effects, supporting the role of autophagy in the disease process

b) Consideration of organ-specific approaches:

• Different compounds may be needed to address muscle versus liver pathology, as drugs that improved muscle phenotypes did not resolve liver pathology

• This suggests potential differences in disease mechanisms between organ systems

c) Cautionary findings:

• Some compounds (particularly wortmannin) worsened the VMA21 mutant phenotype, indicating careful selection of therapeutic candidates is necessary

d) Screening methodology:

• Drug screening can be performed using zebrafish models assessing birefringence (muscle integrity), motor function, and survival as endpoints

• Initial short-term screening followed by long-term assessment in multiple mutant lines provides confirmatory results

Researchers should consider both the direct effects on V-ATPase assembly and the downstream consequences on autophagy when developing therapeutic strategies.

• How can recombinant S. stipitis VMA21 be used to study the assembly mechanism of V-ATPase complexes?

Recombinant S. stipitis VMA21 can be utilized in several experimental approaches to study V-ATPase assembly:

a) Protein-protein interaction studies:

• Co-immunoprecipitation assays to identify binding partners within the V-ATPase complex

• Yeast two-hybrid screening to map interaction domains

• Surface plasmon resonance to measure binding affinities between VMA21 and V-ATPase subunits

b) In vitro reconstitution experiments:

• Cell-free systems to reconstitute V-ATPase assembly with purified components

• Analysis of assembly intermediates using blue native PAGE

• Time-course studies to determine the sequence of assembly events

c) Structural biology approaches:

• Cryo-electron microscopy of VMA21-V-ATPase complexes at different assembly stages

• Cross-linking mass spectrometry to identify proximity relationships

• Hydrogen-deuterium exchange mass spectrometry to identify conformational changes during assembly

d) Functional complementation assays:

• Using recombinant VMA21 to rescue V-ATPase assembly in VMA21-deficient cells

• Structure-function analysis by testing mutant forms of VMA21

Researchers should consider using the VMA21 storage buffer (Tris-based buffer with 50% glycerol) and avoiding repeated freeze-thaw cycles when working with the recombinant protein.

• What is the relationship between VMA21 function and metabolic engineering applications in S. stipitis?

The relationship between VMA21 function and metabolic engineering in S. stipitis involves:

a) Impact on sugar utilization capacity:

• S. stipitis is valued for its capacity to ferment pentose sugars, particularly xylose

• The vacuolar pH maintained by proper V-ATPase function influences sugar transport and metabolism

• Carbon metabolism in S. stipitis differs from S. cerevisiae, with metabolic flux distribution showing higher TCA cycle activity

b) Relevance to biofuel production:

• S. stipitis can produce ethanol from various sugars including xylose, sucrose, glucose, and fructose

• Native strains can produce approximately 50 g/L ethanol in 48h from pure xylose

• The metabolic state influenced by V-ATPase function affects fermentation efficiency

c) Applications in resveratrol production:

• Recombinant S. stipitis has been used for resveratrol production from molasses and other sugar sources

• Vacuolar function may influence the accumulation of metabolic intermediates in the resveratrol biosynthetic pathway

• Intracellular metabolite analysis showed that sugar composition significantly affects metabolite accumulation, including AMP levels

Researchers can apply metabolic flux analysis using 13C-labeled substrates to understand how VMA21 function influences carbon flow through central metabolic pathways, which is critical for metabolic engineering applications .

Technical Methods Questions

• What experimental protocols are recommended for producing high-quality recombinant S. stipitis VMA21 protein?

For high-quality recombinant S. stipitis VMA21 production, the following protocol elements are recommended:

a) Expression system selection:

• Mammalian cell expression systems provide proper folding for membrane proteins

• Use of optimized codon usage for the host system

• Consider inducible expression systems to control protein production levels

b) Purification strategy:

• Use of His-tag for initial affinity purification

• Gentle detergent solubilization (e.g., DDM or CHAPS) to maintain native structure

• Size exclusion chromatography as a final polishing step

c) Buffer optimization:

• Storage in Tris-based buffer with 50% glycerol

• Optimization of pH and salt concentration to maintain stability

• Addition of protease inhibitors to prevent degradation

d) Quality control:

• Purity assessment: aim for >80% purity by SDS-PAGE

• Endotoxin testing: ensure <1.0 EU per μg of protein using LAL method

• Functional validation: assess ability to complement VMA21 deficiency in appropriate cell models

e) Storage recommendations:

• Store at -20°C to -80°C for long-term preservation

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• For short-term storage, maintain at +4°C for up to one week

This protocol framework should be adjusted based on specific research requirements and application context.

• How can researchers quantitatively analyze the impact of VMA21 on cellular metabolism in S. stipitis?

Quantitative analysis of VMA21's impact on S. stipitis metabolism can employ:

a) Metabolic flux analysis:

• 13C-labeled glucose cultivation followed by analysis of proteinogenic amino acids

• Determination of summed fractional labeling (SFL) combined with metabolic flux analysis (MFA)

• Calculation of flux distributions normalized to glucose uptake rate

b) Metabolomics approaches:

• Capillary electrophoresis time-of-flight mass spectrometry for intracellular metabolite analysis

• Gas or liquid chromatography-mass spectrometry for comprehensive metabolite profiling

• Quantification of key metabolites including glycolytic intermediates, TCA cycle intermediates, and ATP/AMP ratios

c) Physiological parameters measurement:

• Determination of specific growth rates, substrate consumption, and product formation rates

• Respiratory quotient assessment to characterize metabolic mode (respiratory vs. fermentative)

• Oxygen uptake rate and carbon dioxide evolution rate measurements

d) Comparative analysis frameworks:

• Comparison between wild-type and VMA21-deficient strains

• Evaluation under different growth conditions (batch vs. chemostat cultivation)

• Statistical methods including Welch's Two Sample t-Test for metabolite comparisons

The table below illustrates a statistical framework for metabolomic data analysis:

This methodological framework allows for comprehensive quantification of VMA21's impact on cellular metabolism .

Research Challenges and Solutions

• What are the key challenges in studying VMA21 function in S. stipitis and how can they be addressed?

Key challenges and potential solutions include:

a) Membrane protein challenges:

• Challenge: Difficulties in expressing and purifying integral membrane proteins like VMA21

• Solution: Use specialized detergents for solubilization; consider fusion partners to enhance stability; optimize buffer conditions with 50% glycerol

b) Functional assessment difficulties:

• Challenge: Directly measuring V-ATPase assembly and activity in vivo

• Solution: Employ LysoTracker staining to assess vacuolar acidification ; use fluorescent protein-tagged V-ATPase subunits to track assembly; measure proton pumping activity in isolated vacuoles

c) Genetic manipulation limitations:

• Challenge: Genetic tools for S. stipitis are less developed than for model yeasts

• Solution: Adapt CRISPR-Cas9 systems for S. stipitis; utilize the genetic toolbox developed for S. stipitis including synthetic drug resistance markers

d) Metabolic complexity:

• Challenge: Distinguishing direct VMA21 effects from secondary metabolic adaptations

• Solution: Use time-course experiments after conditional VMA21 depletion; employ 13C metabolic flux analysis to track metabolic changes ; compare with known V-ATPase inhibitor effects

e) Comparative analysis difficulties:

• Challenge: Making accurate comparisons between S. stipitis and other yeast species

• Solution: Use standardized growth conditions for comparisons ; normalize data to appropriate reference points; employ systems biology approaches integrating multiple data types

These methodological approaches can help overcome technical barriers in studying this challenging but important membrane protein.

• How can researchers investigate the role of VMA21 in autophagy regulation in yeast models compared to mammalian systems?

Investigating VMA21's role in autophagy across yeast and mammalian systems requires:

a) Comparative experimental design:

• Parallel experiments in S. stipitis and mammalian cell models with VMA21 depletion

• Standardized autophagy induction protocols (e.g., nitrogen starvation, rapamycin treatment)

• Use of both genetic and pharmacological approaches to modulate VMA21 function

b) Autophagy markers and monitoring techniques:

• In yeast:

  • GFP-Atg8 processing assay to monitor autophagy flux

  • Electron microscopy to visualize autophagic structures

  • Biochemical assessment of protein degradation rates

• In mammalian systems:

  • LC3I to LC3II conversion monitoring via Western blot

  • GFP-LC3-RFP-LC3ΔG constructs to evaluate autophagic flux in real-time

  • Immunostaining for autophagy markers like LC3 and p62

c) Addressing system-specific differences:

• Account for differences in autophagy machinery between yeast and mammals

• Consider the role of V-ATPase regulation in different cellular compartments

• Explore connections to mTORC1 signaling in mammalian cells versus TOR signaling in yeast

d) Therapeutic implications assessment:

• Test autophagy modulators like edaravone and LY294002 in both systems

• Evaluate potential species-specific differences in drug responses

• Consider combination approaches targeting both V-ATPase assembly and downstream autophagy

This comparative approach provides valuable insights into conserved and divergent aspects of VMA21 function in autophagy regulation across evolutionarily distant systems.