Recombinant Saccharomyces cerevisiae Alkylphosphocholine resistance protein LEM3 (LEM3)

What is the LEM3 protein and what is its primary function in Saccharomyces cerevisiae?

LEM3 (also known as Alkylphosphocholine resistance protein) in Saccharomyces cerevisiae belongs to a conserved family of proteins containing the LEM domain. This protein plays essential roles in membrane biology and DNA processing. In yeast, LEM3 functions primarily in lipid translocation across membranes and contributes to alkylphosphocholine resistance. The protein contains characteristic structural elements including a LEM domain (named after proteins LAP2, Emerin, and MAN1) that facilitates DNA and chromatin interactions .

Similar to its ortholog in C. elegans, yeast LEM3 may participate in DNA repair pathways and potentially possess nuclease activity. Research has shown that LEM3 orthologs in other organisms, such as C. elegans LEM-3, are involved in processing recombination intermediates and contain GIY-YIG nuclease motifs that enable DNA processing activities . Understanding the evolutionary conservation of these functions provides insight into the fundamental roles of LEM3 across species.

How does LEM3 protein structure correlate with its function in DNA processing?

The structure-function relationship of LEM3 proteins reveals important insights into their mechanisms. LEM3 contains several key domains:

• N-terminal LEM domain: Mediates interactions with chromatin through binding to barrier-to-autointegration factor (BAF)

• Ankyrin repeats: Facilitate protein-protein interactions

• GIY-YIG nuclease motif: Enables nucleolytic processing of DNA substrates

These structural elements enable LEM3 to perform its biological functions. For instance, in C. elegans, LEM-3 can cleave supercoiled plasmid DNA into relaxed circular and linear forms, indicating structure-specific endonuclease activity . This activity is related to its ability to process DNA structures with secondary elements, which is crucial for resolving recombination intermediates. Similar structural elements are presumed to be conserved in the yeast ortholog, suggesting parallel functionalities in DNA processing mechanisms.

What are the primary experimental methods to study LEM3 protein expression and localization?

Several methodological approaches are effective for investigating LEM3 expression and localization:

Based on research with LEM-3 in C. elegans, these proteins typically localize between dividing nuclei during cell division, suggesting a role in processing DNA linkages that persist during nuclear division . Similar approaches can be used to determine if yeast LEM3 shows comparable localization patterns during mitosis and meiosis, which would provide insights into its functional conservation across species.

How do mutations in the LEM3 gene affect cellular phenotypes in yeast?

Mutations in LEM3 can lead to distinct phenotypes that reveal its biological functions. Based on comparative studies with related proteins, LEM3 mutations in yeast may result in:

• Increased sensitivity to alkylphosphocholine compounds

• Altered membrane lipid composition and asymmetry

• Defects in DNA damage response pathways

• Synthetic lethality when combined with mutations in other DNA repair genes

Research on LEM-3 in C. elegans has shown that while single mutants may not display overt phenotypes, double mutants with other nucleases like MUS-81 or SLX-1 exhibit 100% embryonic lethality . This suggests redundant pathways in DNA processing where LEM3 provides crucial backup functions. Similar genetic interaction studies in yeast would help elucidate the functional redundancy of LEM3 with other DNA processing enzymes in S. cerevisiae.

How does LEM3 interact with other components of the DNA repair machinery during meiotic recombination?

LEM3 functions within a complex network of DNA repair proteins that process recombination intermediates during meiosis. Based on research in C. elegans, LEM-3 operates in parallel pathways with other nucleases such as MUS-81, SLX-1, and SLX-4 . The interaction network includes:

• Direct physical interactions: LEM3 may form protein complexes with other repair factors

• Pathway redundancy: LEM3 provides backup functions when primary resolution pathways fail

• Temporal coordination: LEM3 activity may be regulated to occur at specific stages of meiosis

Research has shown that in C. elegans, LEM-3 and MUS-81 act in conjunction to process early recombination intermediates in meiosis . When both are absent, there are alterations in recombination marker profiles and delays in the processing of crossover designation markers. Similar interaction patterns likely exist in yeast, where LEM3 may work with the yeast homologs of these proteins to ensure complete resolution of DNA intermediates during meiosis.

Methodologically, researchers should employ co-immunoprecipitation, yeast two-hybrid screening, and genetic interaction studies to fully characterize these interactions in S. cerevisiae.

What is the enzymatic mechanism by which LEM3 resolves DNA recombination intermediates?

LEM3 belongs to the GIY-YIG nuclease family, which provides insights into its likely enzymatic mechanism. Based on research with its orthologs, LEM3 likely functions through the following mechanism:

• DNA substrate recognition: Preferential binding to structured DNA intermediates

• Nucleophilic attack: The active site coordinates metal ions to facilitate cleavage

• Phosphodiester bond hydrolysis: Breaking the DNA backbone at specific sites

• Product release: Generating processed DNA structures

Studies of LEM-3 in C. elegans have demonstrated its ability to cleave supercoiled plasmid DNA and substrates rich in secondary structures . This suggests that yeast LEM3 may similarly recognize and process structured DNA intermediates that arise during recombination. The nuclease likely has specificity for particular DNA structures rather than sequences, allowing it to target aberrant DNA conformations that arise during recombination.

Researchers investigating the enzymatic mechanism should employ in vitro nuclease assays with purified recombinant protein and various DNA substrates to determine structure preferences and catalytic parameters.

How does post-translational modification regulate LEM3 activity during the cell cycle?

The activity of LEM3 is likely regulated through various post-translational modifications (PTMs) that coordinate its function with cell cycle progression:

Similar to YEN1/GEN1 nucleases in yeast, which show cell cycle-dependent regulation of enzymatic activity , LEM3 activity may be kept at low levels during prophase but induced during later stages of cell division. This regulation would ensure that LEM3 serves as a safeguard activity that only processes DNA linkages that escape the attention of primary resolution pathways.

Methodologically, researchers should use phospho-specific antibodies, mass spectrometry, and site-directed mutagenesis of PTM sites to characterize how these modifications affect LEM3 function throughout the cell cycle.

What are the implications of LEM3 dysfunction for genome stability and chromosome segregation?

LEM3 dysfunction has significant consequences for genome maintenance, particularly during meiosis and mitosis:

• Persistent DNA linkages: When LEM3 is absent, unresolved recombination intermediates may persist into cell division

• Chromosome segregation defects: These linkages can impair proper chromosome separation

• Aneuploidy: Improper segregation can lead to chromosome number abnormalities

• Increased mutation rates: Failed repair may lead to genomic instability

Research in C. elegans has shown that depletion of LEM-3 leads to accumulation of chromosome linkages, especially during meiosis II, indicating that LEM-3 directly processes DNA linkages caused by unresolved recombination intermediates . The protein localizes between dividing nuclei during meiotic division, suggesting a direct role in resolving persistent DNA bridges that would otherwise prevent proper chromosome segregation.

To study these implications in yeast, researchers should employ live-cell imaging of chromosome segregation, pulse-field gel electrophoresis to assess chromosome integrity, and genetic assays to measure recombination and mutation rates in LEM3-deficient strains.

How do environmental stressors affect LEM3 expression and activity in Saccharomyces cerevisiae?

Environmental stressors likely modulate LEM3 expression and activity to enhance cellular resilience:

• DNA damaging agents: May upregulate LEM3 to increase DNA repair capacity

• Oxidative stress: Can alter protein function through oxidation of sensitive residues

• Temperature shock: May affect protein folding and enzymatic activity

• Nutrient limitation: Could change expression patterns through stress-response pathways

Studies in C. elegans have shown that LEM-3 mutants are hypersensitive to ionizing irradiation, UV treatment, and DNA cross-linking agents , indicating that LEM3 plays important roles in responding to DNA damage. Similar sensitivity profiles might be observed in yeast lacking functional LEM3, particularly under conditions that increase the formation of recombination intermediates.

Researchers should investigate LEM3 expression under different stress conditions using RT-qPCR, RNA-seq, and proteomics approaches, while functional studies should employ sensitivity assays to various DNA damaging agents in wild-type versus LEM3-deficient strains.

What are the optimal conditions for expressing and purifying recombinant LEM3 protein for biochemical studies?

Successful expression and purification of functional recombinant LEM3 requires careful optimization:

For optimal expression:

• Use codon-optimized sequences for the expression host

• Express LEM3 domains separately if full-length protein shows poor solubility

• Include protease inhibitors during purification to prevent degradation

• Test multiple buffer conditions to maintain stability and activity

• Verify nuclease activity with model substrates after purification

The GIY-YIG nuclease domain should be expressed with particular care, as improper folding may lead to loss of enzymatic activity. Researchers should verify the structural integrity of purified protein using circular dichroism spectroscopy and assess enzymatic function through nuclease activity assays with model DNA substrates.

How can CRISPR-Cas9 genome editing be optimized for studying LEM3 function in Saccharomyces cerevisiae?

CRISPR-Cas9 provides powerful approaches for investigating LEM3 function through precise genetic modifications:

• Guide RNA design:

  • Target unique sequences to avoid off-target effects

  • Use validated S. cerevisiae CRISPR tools with optimized promoters

  • Design guide RNAs with minimal secondary structures

• Repair template design:

  • Include at least 40bp homology arms for efficient homologous recombination

  • Introduce silent mutations in the PAM site to prevent re-cutting

  • Consider adding reporter tags (GFP, FLAG) to track protein expression and localization

• Validation strategies:

  • Confirm edits by PCR and sequencing

  • Verify protein expression through Western blotting

  • Assess localization using fluorescence microscopy

  • Test functional consequences using phenotypic assays

For domain function studies, researchers should create precise mutations in the GIY-YIG nuclease domain to disrupt enzymatic activity while preserving protein structure. Complementation studies with wild-type and mutant variants can reveal the importance of specific residues for LEM3 function in vivo.

What high-throughput screening approaches can identify novel genetic interactions with LEM3?

Several high-throughput approaches can effectively uncover genetic interactions with LEM3:

• Synthetic Genetic Array (SGA) analysis:

  • Cross LEM3 deletion strain with genome-wide deletion collection

  • Score growth phenotypes to identify synthetic lethal/sick interactions

  • Validate candidates with targeted crosses and tetrad analysis

• Barcode-based pooled screens:

  • Create barcoded LEM3 mutant strain collections

  • Culture pooled strains under different conditions

  • Use next-generation sequencing to identify depleted/enriched barcodes

• CRISPR interference screens:

  • Deploy genome-wide sgRNA libraries in LEM3-deficient background

  • Identify genes whose depletion affects viability in LEM3 mutants

  • Validate hits with individual strains and complementation tests

Studies in C. elegans have identified synthetic lethal interactions between LEM-3 and other nucleases like SLX-1 and MUS-81 . Similar approaches in yeast would reveal whether LEM3 functions in parallel DNA processing pathways, providing insights into the conservation of these genetic interactions across species.

How can advanced imaging techniques be applied to study LEM3 dynamics during DNA recombination and repair?

Advanced imaging approaches offer powerful tools for visualizing LEM3 function in living cells:

• Super-resolution microscopy:

  • PALM/STORM imaging can achieve ~20nm resolution

  • Structured illumination microscopy (SIM) provides 3D resolution enhancement

  • Stimulated emission depletion (STED) microscopy reveals fine localization details

• Live-cell imaging applications:

  • Track LEM3-fluorescent protein fusions during cell cycle progression

  • Use photoactivatable fluorescent proteins to monitor protein movement

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure protein dynamics

• Multi-color imaging strategies:

  • Simultaneously visualize LEM3 with DNA damage markers

  • Co-localize with recombination intermediates and repair factors

  • Track chromosome dynamics during segregation

In C. elegans, LEM-3 localizes between dividing meiotic nuclei , suggesting it processes DNA linkages during nuclear division. Similar imaging approaches in yeast would reveal whether LEM3 shows comparable dynamic localization patterns during mitosis and meiosis, providing insights into its role in maintaining genome stability during cell division.

How does LEM3 contribute to lipid asymmetry maintenance in yeast membranes?

Beyond its role in DNA processing, LEM3 plays critical functions in membrane biology:

• Phospholipid translocation:

  • Facilitates movement of phospholipids between membrane leaflets

  • Maintains phosphatidylethanolamine asymmetry

  • Influences membrane fluidity and integrity

• Interaction with flippase complexes:

  • Functions with Dnf1/Dnf2 P4-ATPases

  • Enables proper trafficking of membrane components

  • Required for efficient lipid transport activity

• Consequences for membrane-dependent processes:

  • Endocytosis and vesicular trafficking

  • Signaling pathway compartmentalization

  • Stress response and adaptation

This membrane-related function appears distinct from its DNA processing role, suggesting LEM3 has evolved dual functionalities. Researchers should investigate whether these functions are mechanistically linked or represent independent roles that have converged in the same protein through evolution.

What is the evolutionary relationship between LEM3 in different species and how has its function diversified?

Evolutionary analysis reveals important insights about LEM3 functional conservation and specialization:

• Phylogenetic distribution:

  • LEM3/Ankle1 is conserved specifically in animals and fungi

  • Different domains show varying degrees of conservation

  • Nuclease motifs are highly conserved across species

• Functional diversification:

  • Membrane functions predominate in some lineages

  • DNA processing roles are emphasized in others

  • Some species utilize both functions

• Structural adaptations:

  • Domain architecture variations between species

  • Species-specific regulatory elements

  • Lineage-specific interacting partners

In C. elegans, LEM-3/Ankle1 contains an N-terminal LEM domain, Ankyrin repeats, and a GIY-YIG nuclease motif . This nuclease motif is also found in bacterial UvrC nucleotide excision repair proteins and in the distantly related SLX1 nuclease, suggesting ancient evolutionary origins for this functional domain.

How can understanding LEM3 function contribute to developing novel biotechnological applications?

Knowledge of LEM3 function opens possibilities for biotechnological innovations:

• DNA processing applications:

  • Development of structure-specific nucleases for genome engineering

  • Tools for selective DNA modification in synthetic biology

  • Novel approaches for manipulating recombination outcomes

• Membrane engineering applications:

  • Systems for controlling membrane composition in bioprocessing

  • Enhanced stress tolerance in industrial yeast strains

  • Improved production of membrane-associated compounds

• Disease-relevant applications:

  • Insights into pathologies caused by defective DNA processing

  • Potential therapeutic targets for diseases with aberrant recombination

  • Models for studying genomic instability disorders

Understanding the molecular mechanisms of LEM3 function provides foundational knowledge that can be translated into practical applications across multiple fields of biotechnology and biomedicine.