Recombinant Saccharomyces cerevisiae Probable undecaprenyl pyrophosphate synthase (NUS1)

What is NUS1 in Saccharomyces cerevisiae and what is its relevance to scientific research?

NUS1 (Nuclear Undecaprenyl Pyrophosphate Synthase 1) in Saccharomyces cerevisiae encodes a membrane protein that functions as a probable undecaprenyl pyrophosphate synthase. According to protein databases, it is also known as UPP synthase (EC 2.5.1.31), di-trans,poly-cis-decaprenylcistransferase, or undecaprenyl pyrophosphate synthase . The protein is encoded by the YDL193W locus in the yeast genome.

The scientific importance of NUS1 extends beyond basic yeast biology to translational medicine, as homologs of this gene exist in humans and have been implicated in neurological disorders, including Parkinson's disease. The yeast NUS1 serves as a valuable model for understanding the fundamental mechanisms of this protein family across species and provides insights into the pathogenesis of human diseases associated with NUS1 dysfunction .

Research on NUS1 contributes to our understanding of dolichol biosynthesis and protein glycosylation pathways, which are essential cellular processes conserved from yeast to humans. The ability to produce and study recombinant forms of this protein enables detailed structural and functional analyses that may inform therapeutic development for NUS1-related disorders.

What is the molecular structure and functional domains of the NUS1 protein?

The NUS1 protein in Saccharomyces cerevisiae is a full-length protein consisting of 375 amino acids. The amino acid sequence includes several functional domains critical for its enzymatic activity as an undecaprenyl pyrophosphate synthase. The complete amino acid sequence is documented in protein databases (UniProt NO.: Q12063) .

The protein structure includes transmembrane regions, as suggested by the presence of hydrophobic amino acid stretches within the sequence. The functional domains likely include catalytic sites for the enzymatic activity that facilitates the synthesis of undecaprenyl pyrophosphate, a critical intermediate in various cellular biosynthetic pathways.

Structural analysis suggests that the protein contains regions responsible for membrane anchoring, substrate binding, and catalytic activity. The conservation of certain amino acid residues across species indicates their essential role in protein function. Researchers studying NUS1 should pay particular attention to these conserved regions when designing mutational studies or when analyzing the potential functional impact of naturally occurring variants.

How is recombinant NUS1 protein typically produced for research applications?

Production of recombinant NUS1 protein for research typically involves expression systems optimized for membrane proteins. The general methodology includes:

• Cloning: The NUS1 gene (YDL193W from S. cerevisiae) is amplified and cloned into an appropriate expression vector containing necessary regulatory elements and affinity tags for purification.

• Expression System Selection: Researchers must choose between prokaryotic (E. coli) or eukaryotic expression systems (yeast, insect cells, mammalian cells). For functional studies requiring proper folding and post-translational modifications, eukaryotic expression systems are often preferred.

• Protein Expression: Controlled expression conditions are established, typically involving induction at specific growth phases and optimized temperature, pH, and media composition.

• Extraction and Purification: Given NUS1's membrane-associated nature, specialized detergent-based extraction methods are employed, followed by affinity chromatography using the incorporated tags. Additional purification steps may include ion-exchange chromatography or size-exclusion chromatography.

• Storage and Stability: The purified protein is stored in appropriate buffer conditions, often with 50% glycerol as indicated in commercial preparations, to maintain stability at -20°C or -80°C for extended storage .

Commercial preparations typically provide the protein at concentrations suitable for experimental applications (e.g., 50 μg) in optimized storage buffers that maintain protein stability and activity . It is recommended to avoid repeated freeze-thaw cycles and to store working aliquots at 4°C for up to one week to preserve functionality.

What is the relationship between yeast NUS1 and human NUS1, particularly in the context of Parkinson's disease research?

The relationship between yeast and human NUS1 provides a powerful translational research model. Human NUS1 encodes the Nogo-B receptor (NgBR), which is required for dolichol biosynthesis and protein glycosylation, functions that appear conserved with its yeast counterpart . This functional homology makes S. cerevisiae NUS1 a valuable model for studying fundamental aspects of NUS1 biology that may be relevant to human disease.

Further literature review covering 5,142 PD cases (including at least 116 early-onset PD cases) from 59 articles revealed no pathogenic or disease-associated NUS1 variants . Another study using direct sequencing of full coding regions and exon-intron boundaries in 494 sporadic PD cases also showed negative results .

These contradictory findings highlight the complex nature of genetic contributions to PD and demonstrate the importance of:

• Comprehensive genetic screening approaches

• Careful statistical analysis of variant frequencies

• Functional validation of potential disease-associated variants

• Consideration of population-specific genetic architecture

Researchers using yeast NUS1 as a model for human disease should be aware of these nuances and design experiments that can provide functional insights applicable across species.

What experimental approaches have been used to study the role of NUS1 in genome plasticity and DNA amplification?

Research has revealed that NUS1 can be involved in eccDNA (extrachromosomal circular DNA element) formation and gene amplification, representing important mechanisms of genome plasticity in Saccharomyces cerevisiae. While most DNA amplifications in yeast typically involve pre-existing repetitive sequences like ribosomal DNA, Ty elements, or Long Terminal Repeats (LTRs), studies have documented the generation of eccDNA in regions without repetitive sequences during adaptive evolution experiments .

Key experimental approaches to study NUS1's role in genome plasticity include:

• Adaptive Evolution Experiments: Researchers subject yeast to selective pressure that favors amplification of specific genes, then perform whole genome sequencing to identify amplification events .

• Comparative Genomics: Whole genome sequence comparison between evolved strains and parent strains can reveal amplification events. For example, research has identified cases where heterologous genes inserted near ARS (Autonomously Replicating Sequence) elements underwent significant amplification during adaptive evolution .

• Temporal Analysis: Monitoring the amplification process during adaptive evolution by sampling at different time points allows researchers to track the formation and dynamics of eccDNA elements .

• Molecular Characterization of eccDNA: Techniques such as inverse PCR, Southern blotting, and next-generation sequencing can be employed to characterize the structure, copy number, and sequence features of eccDNA containing NUS1 or other genes.

• Functional Validation: Genetic approaches including gene knockouts, complementation studies, and site-directed mutagenesis help determine the functional significance of NUS1 amplification or the role of NUS1 in facilitating the amplification of other genes.

These approaches have provided insights into the mechanisms of genome plasticity in yeast and may have implications for understanding similar processes in higher eukaryotes, including humans.

How does the function of NUS1 relate to cellular pathways implicated in neurodegenerative disorders?

NUS1 functions in several cellular pathways that are increasingly recognized as important in neurodegenerative disorders, particularly Parkinson's disease. Understanding these connections provides valuable insights for researchers investigating disease mechanisms.

The NUS1 protein is required for dolichol biosynthesis and protein glycosylation . These processes are fundamentally important for proper protein folding, trafficking, and function. Disruptions in glycosylation pathways have been implicated in various neurodegenerative disorders through mechanisms including:

• Protein Misfolding and Aggregation: Improper glycosylation can lead to protein misfolding, a hallmark of many neurodegenerative diseases including PD where α-synuclein aggregation plays a central role.

• Endoplasmic Reticulum (ER) Stress: Defects in glycosylation can trigger ER stress and activate the unfolded protein response, pathways implicated in neurodegeneration.

• Mitochondrial Dysfunction: Some research suggests connections between glycosylation pathways and mitochondrial function, which is prominently affected in PD.

• Synaptic Function: Proper glycosylation is essential for many synaptic proteins, and synaptic dysfunction is an early feature of several neurodegenerative disorders.

Interestingly, homozygous NUS1 mutations in humans cause congenital disorder of glycosylation type Iaa (CDG1AA, OMIM 617082), while rare heterozygous mutations have been reported in cases with autosomal dominant mental retardation-55 with seizures (MRD55, OMIM 617831) . These established neurological phenotypes further support the potential relevance of NUS1 to brain function and neurodegeneration.

What are the critical factors to consider when designing genetic studies to investigate NUS1 variants in disease?

Designing robust genetic studies to investigate NUS1 variants in disease requires careful consideration of multiple methodological factors. Based on published research approaches, researchers should consider:

• Cohort Selection and Size:

  • Include both sporadic and familial cases of the disease

  • Ensure adequate representation of early-onset cases, which may have stronger genetic components

  • Match controls ethnically to minimize population stratification effects

  • Power calculations should guide sample size determination

• Sequencing Approach:

  • Whole exome sequencing provides comprehensive coverage of coding regions and exon-intron boundaries

  • Consider high-coverage next-generation sequencing for regions of particular interest

  • Validate identified variants using orthogonal methods (e.g., Sanger sequencing)

• Variant Analysis Pipeline:

  • Annotate variants using multiple population databases (1,000 Genomes Project, ExAC, gnomAD)

  • Employ multiple bioinformatic prediction tools to assess potential functional impact (e.g., MutationTaster, SIFT, PROVEAN, PANTHER, PolyPhen-2)

  • Check Hardy-Weinberg equilibrium in control groups

  • Perform appropriate statistical tests to identify frequency biases between cases and controls

• Family Studies:

  • When possible, perform segregation analysis in families to strengthen evidence for pathogenicity

  • The absence of co-segregation data significantly weakens claims about disease causation

• Exclusion of Known Genetic Causes:

  • Screen for and exclude known disease-causing mutations in established genes

  • Document details of this exclusion process in publications

The importance of these considerations is highlighted by critical evaluations of previous research. For example, one study noted several limitations in prior work claiming NUS1 as a PD-causing gene, including lack of co-segregation evidence, absence of screening for other genetic causes, and unavailability of relatives for variant confirmation .

What protocol optimizations are recommended for expressing and purifying functional recombinant NUS1 protein?

Expressing and purifying functional recombinant NUS1 protein presents several challenges due to its membrane-associated nature. Based on available literature and standard practices for similar proteins, the following protocol optimizations are recommended:

Expression System Selection:

• For basic structural studies: E. coli systems with specialized tags (e.g., MBP, SUMO) to enhance solubility

• For functional studies: S. cerevisiae expression systems that provide native folding environment and post-translational modifications

• For detailed structural analysis: Consider Pichia pastoris or insect cell systems that combine high yield with eukaryotic processing

Vector Design Considerations:

• Include a cleavable affinity tag (His6, GST, or FLAG) for purification

• Consider fusion partners that enhance membrane protein expression and solubility

• Include a TEV or PreScission protease cleavage site for tag removal after purification

Expression Optimization:

• Temperature: Lower temperatures (16-20°C) often improve proper folding

• Induction: Gentle induction with lower inducer concentrations for longer periods

• Media supplements: Consider additives that stabilize membrane proteins

Extraction and Solubilization:

• Screen multiple detergents for optimal extraction (n-dodecyl-β-D-maltoside, digitonin, CHAPS)

• Consider nanodisc or amphipol technologies for maintaining native-like environment

• Optimize detergent:protein ratios to maintain structural integrity

Purification Strategy:

• Two-step purification minimum: affinity chromatography followed by size-exclusion chromatography

• Buffer optimization: Inclusion of glycerol (e.g., 50%) and appropriate salt concentration as indicated in commercial preparations

• Consider lipid supplementation during purification to maintain structural stability

Storage Conditions:

• Store at -20°C for regular use, or -80°C for extended storage

• Avoid repeated freeze-thaw cycles as recommended for commercial preparations

• Prepare working aliquots that can be stored at 4°C for up to one week

Functional Validation:

• Develop activity assays specific to undecaprenyl pyrophosphate synthase function

• Verify proper folding using circular dichroism or limited proteolysis

• For structural studies, assess protein homogeneity using dynamic light scattering

Implementation of these optimizations should be approach methodically, with systematic testing of variables to identify the conditions that yield the highest quality functional protein for the intended research application.

What are the best approaches for functional validation of NUS1 variants identified in genetic studies?

Functional validation of NUS1 variants is essential for establishing their pathogenicity and understanding their mechanistic contributions to disease. Based on current research methodologies, the following comprehensive approach is recommended:

In Silico Analysis:

• Apply multiple prediction algorithms to assess potential impact on protein structure/function

• Perform molecular modeling to visualize how variants might affect protein folding, binding sites, or catalytic regions

• Analyze conservation across species to identify evolutionarily constrained residues

Yeast Models:

• Leverage S. cerevisiae as a model system for variant testing via:

  • Knockout/complementation studies: Delete native NUS1 and complement with variant forms

  • Growth assays under various stress conditions to reveal subtle phenotypes

  • Double mutant analyses with genetically interacting partners

Biochemical Characterization:

• Express and purify wild-type and variant proteins to compare:

  • Enzymatic activity using specific assays for undecaprenyl pyrophosphate synthase function

  • Protein stability through thermal shift assays or limited proteolysis

  • Binding properties with interaction partners or substrates

  • Structural changes using circular dichroism or other biophysical techniques

Cellular Models:

• Assess variant effects in both yeast and mammalian cell systems:

  • Protein localization using fluorescent tagging or subcellular fractionation

  • Effects on dolichol biosynthesis and protein glycosylation pathways

  • Impacts on cellular stress responses (ER stress, unfolded protein response)

  • Influence on mitochondrial function (membrane potential, morphology, respiration)

Mammalian Disease-Relevant Assays:

• For variants potentially linked to neurodegenerative disease:

  • Effects on neuronal survival, morphology, and function

  • Interaction with disease-related proteins (e.g., α-synuclein for PD-related studies)

  • Impact on key cellular pathways implicated in neurodegeneration

Integration with Pathways Analysis:

• Examine effects on main pathobiological pathways implicated in relevant diseases:

  • Autophagy

  • Endocytosis

  • Mitochondrial biology

  • Immune response

  • Lysosomal function

What are the most promising future research directions for NUS1 in both basic science and translational applications?

Future research on NUS1 holds significant promise for advancing both fundamental understanding of cellular processes and translational applications in medicine. Several key research directions deserve particular attention:

Basic Science Directions:

• Detailed structural determination of the NUS1 protein through X-ray crystallography or cryo-EM to elucidate its molecular mechanism

• Comprehensive characterization of NUS1's role in dolichol biosynthesis and the regulatory mechanisms controlling this pathway

• Exploration of NUS1's potential functions beyond glycosylation pathways, particularly in membrane dynamics and cellular stress responses

• Investigation of the evolutionary conservation of NUS1 function across species and potential species-specific adaptations

Genomic Stability and Adaptation:

• Further examination of NUS1's involvement in eccDNA formation and gene amplification events during adaptive evolution

• Analysis of how NUS1-related genomic plasticity contributes to cellular adaptation to environmental stresses

• Development of systems to monitor and manipulate NUS1-mediated genomic changes in real-time

Disease Mechanisms:

• Clarification of the actual role of NUS1 variants in Parkinson's disease through larger cohort studies with particular focus on familial PD and early-onset PD

• Exploration of potential NUS1 involvement in other neurodegenerative disorders through similar genetic approaches

• Investigation of the functional interplay between NUS1 and established disease-causing genes in neurodegenerative disorders

Therapeutic Applications:

• Development of small molecule modulators of NUS1 function for potential therapeutic applications

• Exploration of gene therapy approaches for congenital disorders of glycosylation related to NUS1 dysfunction

• Investigation of NUS1 as a potential biomarker for disease susceptibility or progression

Methodological Advancements:

• Refinement of heterologous expression systems for producing high-quality recombinant NUS1 for structural and functional studies

• Development of high-throughput screening methods for NUS1 variant functional characterization

• Creation of advanced cellular and animal models with precise NUS1 mutations that recapitulate human disease features