Recombinant Saccharomyces cerevisiae Seipin FLD1 (FLD1)

What is Saccharomyces cerevisiae Seipin FLD1 and what is its significance in lipid droplet research?

Saccharomyces cerevisiae Seipin FLD1 (also known as YLR404W or Sei1) is a yeast gene encoding a functional homologue of human seipin, originally identified in a screen of approximately 4,700 S. cerevisiae mutants for abnormalities in lipid droplet (LD) number and morphology . FLD1 plays a crucial role in regulating lipid droplet biogenesis, size, and morphology at the endoplasmic reticulum (ER). The significance of FLD1 in research extends beyond basic yeast biology, as mutations in human seipin cause Berardinelli-Seip congenital lipodystrophy (BSCL) and certain motoneuron disorders . Studying FLD1 provides mechanistic insights into how seipin and its homologues modulate the formation and fusion of lipid droplets, offering a valuable model system for understanding the molecular basis of lipodystrophy.

What phenotypes are observed in FLD1 deletion mutants?

FLD1 deletion (fld1Δ) results in distinct abnormal lipid droplet phenotypes:

• Supersized lipid droplets: A fraction of fld1Δ cells display fewer but abnormally enlarged LDs .

• Clustered small irregular droplets: Many fld1Δ cells show clusters of small irregular droplets with electron-dense inclusions entangled in locally proliferated ER ("LD–ER tangles") .

These phenotypes can be modulated by inositol, a phospholipid precursor. At low inositol concentrations, supersized LDs are more prevalent, while high inositol concentrations increase the frequency of LD aggregates . Unlike similar morphological defects seen in other mutants (e.g., opi3Δ), the abnormal LDs in fld1Δ cells are not rescued by stimulation of the Kennedy pathway, indicating a distinct mechanistic defect .

How do FLD1 mutants affect lipid droplet biogenesis?

FLD1/seipin specifically influences the initiation of lipid droplet formation. In chromosomal systems where triacylglycerol synthesis is induced, only 15% of seipin-deficient cells (3KO(GALDGA1)fld1Δ) produce lipid droplets after 3 hours, increasing to only 27% after 9 hours, compared to 82% and 98% respectively in control cells . When droplets do form in mutant cells, they are fewer in number and display aberrant morphology. This biogenetic defect occurs regardless of which triacylglycerol-producing enzyme (Dga1 or Lro1) is used, indicating that seipin's effect is not linked to a specific acyltransferase but rather to the general process of lipid droplet formation .

What is the molecular structure of Sei1/FLD1 protein?

Cryo-electron microscopy has revealed that yeast Sei1 assembles into a homooligomeric ring structure with a resolution of 2.7Å . Key structural features include:

• A decameric arrangement composed of 10 Sei1 protomers

• Outer ring diameter of 140Å and inner ring diameter of 25Å

• Each protomer contains an 8-strand β-sandwich capped by two orthogonal short helices (α1 and α2) at the inner surface

• The β-sandwich fold resembles that of cholesterol-binding protein NPC2

This ring structure appears to be a conserved feature across species, though the number of protomers varies between organisms, with human seipin forming an 11-mer and fly seipin forming a 12-mer .

How does the Sei1-Ldb16 complex function mechanistically in lipid droplet formation?

The Sei1-Ldb16 complex in yeast serves as a molecular machinery for lipid droplet formation through several coordinated functions:

• Unlike human seipin, yeast Sei1 alone cannot concentrate triacylglycerol (TAG)

• Sei1 positions Ldb16, which concentrates TAG within the Sei1 ring through critical hydroxyl residues

• Sei1 transmembrane segments (TMs) promote TAG recruitment to the complex and control Ldb16 stability

The complex acts as a diffusion barrier at ER-LD contact sites, preventing equilibration of ER and LD surface components, thereby maintaining LD identity during biogenesis . In the absence of this complex, phospholipid packing defects occur, leading to aberrant distribution of lipid-binding proteins and abnormal LDs . This mechanism involves sequential TAG-concentrating steps via distinct elements in the ER membrane and lumen .

What critical domains in Sei1/FLD1 are essential for its function?

Several domains in Sei1/FLD1 are crucial for proper function:

Interestingly, human seipin TMs can functionally replace yeast Sei1 TMs, suggesting evolutionary conservation of TAG-binding capacity despite sequence differences .

How can recombinant FLD1/Sei1 be expressed and purified for structural studies?

For structural studies of Sei1/FLD1, researchers have successfully employed the following protocol:

• Express C-terminally tagged Sei1 (Sei1-FLAG) using a galactose-inducible promoter in S. cerevisiae

• Isolate crude membranes and solubilize using dodecyl maltoside (DDM) supplemented with cholesterol hemisuccinate (CHS)

• Perform affinity purification of Sei1-FLAG followed by size-exclusion chromatography

• Verify homogeneity of particles using negative stain electron microscopy before proceeding to cryo-EM analysis

This approach yielded samples suitable for high-resolution (2.7Å) structural determination, revealing the decameric organization of the Sei1 complex .

What imaging techniques are most effective for studying FLD1-related lipid droplet phenotypes?

Multiple complementary imaging approaches are used to characterize FLD1-related phenotypes:

For example, studies have used BODIPY staining to demonstrate that FBs (lipid droplets) in 4KOfld1Δ strains were less intense with less distinct borders, accompanied by enhanced membrane staining .

What genetic approaches can distinguish between the roles of FLD1/Sei1 and Ldb16?

Several genetic strategies have been employed to dissect the distinct roles of Sei1 and Ldb16:

• Generation of single and double deletion mutants (sei1Δ, ldb16Δ, sei1Δldb16Δ) to compare phenotypes

• Complementation studies with plasmid-expressed wild-type or mutant proteins

• Domain swap experiments, such as Sei1hsTM (yeast Sei1 luminal domain with human seipin TMs)

• Expression of human seipin in sei1Δldb16Δ cells to test functional conservation

• Creation of chromosomal systems with inducible triacylglycerol synthesis (e.g., 3KO(GALDGA1))

These approaches have revealed that while both proteins are required for normal LD formation, they have distinct molecular functions - Sei1 forms the oligomeric ring scaffold while Ldb16 concentrates TAG within this ring .

How does phospholipid metabolism influence FLD1-dependent lipid droplet morphology?

The relationship between phospholipid metabolism and FLD1-dependent LD morphology is complex and not fully understood. Key observations include:

• Inositol concentration dramatically affects LD phenotypes in fld1Δ cells, with low inositol favoring supersized LDs and high inositol increasing LD aggregates

• Despite this phenotypic connection to phospholipid precursors, consistent global changes in phospholipid composition have not been detected in seipin mutants

• Abnormal distribution of phosphatidic acid (PA) may occur in seipin mutants, as evidenced by the relocalization of PA sensors like GFP-Spo20 51-91

• Opi1-GFP, which binds PA, forms abnormal foci in ldb16Δ cells, suggesting altered lipid distribution

These findings suggest that FLD1 may influence local phospholipid organization rather than global composition, potentially affecting the physical properties of the LD monolayer and ER-LD contact sites .

What mechanistic models explain how the Sei1/Ldb16 complex prevents abnormal lipid droplet fusion?

Current research suggests several potential mechanisms by which the Sei1/Ldb16 complex prevents abnormal LD fusion:

• Diffusion barrier model: The complex acts as a diffusion barrier at ER-LD contact sites, maintaining distinct membrane compositions between the organelles

• Phospholipid organization: By regulating phospholipid packing at the LD surface, the complex may prevent conditions that favor spontaneous fusion

• Sequential TAG concentration: The stepwise process of TAG concentration within the Sei1 ring, facilitated by Ldb16, may ensure controlled LD growth rather than fusion of existing droplets

• Stabilization of ER-LD contacts: Proper organization of these contact sites may prevent inappropriate interactions between adjacent LDs

Lipid droplets from fld1Δ cells show significantly enhanced fusion activities both in vivo and in vitro, supporting the critical role of FLD1 in preventing excessive LD fusion .

How do experimental findings from yeast FLD1 translate to understanding human seipin-related lipodystrophy?

Research on yeast FLD1 has provided several insights relevant to human seipin-related disorders:

• Expression of human seipin rescues LD-associated defects in fld1Δ cells, demonstrating functional conservation despite limited sequence homology

• Both yeast and human cells lacking seipin accumulate small abnormal droplets, suggesting conserved mechanisms of LD regulation

• The organization of the oligomeric ring structure is preserved across species, though with different numbers of protomers

• Unlike yeast Sei1, human seipin does not require a separate binding partner (like Ldb16), suggesting it incorporates all necessary functionalities

These findings suggest that the fundamental mechanisms of seipin function in LD biogenesis are conserved, though human seipin may have evolved additional roles related to adipocyte development and function that are absent in yeast . Understanding these conserved and divergent aspects provides a framework for investigating how seipin mutations lead to lipodystrophy in humans.

What factors affect the reproducibility of FLD1-related phenotypes in experimental systems?

Several factors can influence the reproducibility of FLD1-related phenotypes:

• Inositol concentration in growth media dramatically affects the distribution of supersized versus aggregated LD phenotypes

• The choice of experimental system (plasmid-based versus chromosomal expression) impacts the severity of LD biogenetic defects

• The timing of observation after induction of TAG synthesis is critical, with phenotypic differences becoming more pronounced at later timepoints

• The specific genetic background used (e.g., which other genes involved in TAG synthesis are present or deleted) affects the manifestation of phenotypes

• Methods of lipid droplet visualization and quantification can influence the interpretation of results

Researchers should carefully control these variables and clearly report experimental conditions to ensure reproducibility across studies.

How can contradictory data regarding FLD1 function be reconciled in the research literature?

The FLD1 literature contains some apparent contradictions that can be reconciled through careful analysis:

• The initial naming of FLD1 as "few lipid droplets" may seem contradictory to the observation that deletion results in both supersized LDs and LD aggregates. This can be reconciled by understanding that early fluorescence screens identified fewer distinct droplets, while later studies with improved imaging revealed the complexity of the phenotype

• While early studies reported phospholipid alterations in fld1Δ cells, later work found no consistent global changes in phospholipid composition. This apparent contradiction may be resolved by focusing on local rather than global lipid changes

• Different studies report varying severities of LD biogenetic defects in seipin-deficient cells. These differences likely arise from variations in experimental systems, with chromosomal expression systems showing more pronounced defects than plasmid-based systems

By considering these methodological differences, researchers can better interpret seemingly contradictory findings in the literature.

What experimental design considerations are crucial when studying FLD1 interactions with other proteins?

When investigating FLD1 interactions with binding partners, researchers should consider:

These considerations help ensure that experimental findings accurately reflect the biological reality of FLD1 interactions.