Screening of genes involved in Lipid Droplet morphology and inheritance in Saccharomyces cerevisiae

Abstract

Lipid droplets (LDs) are conserved organelles that store neutral lipids and are essential for energy metabolism, lipid homeostasis, and stress responses. Although their formation and metabolic regulation have been extensively studied, the mechanisms that govern their inheritance and morphological integrity during cell division are poorly understood. To identify non-essential genes involved in LD inheritance and morphology, I conducted a fluorescence microscopy-based screen using Saccharomyces cerevisiae, examining 400 single gene deletion strains. LDs were visualized by BODIPY 493/503 staining, and their phenotypes were classified as inheritance defects based on LD presence in daughter cells, or as morphological abnormalities based on changes in droplet number, size, or shape. Among the screened strains, deletion of LDB16, a component of the seipin complex, resulted in a clear LD inheritance defect and an increase in LD size. In this mutant strain, 52% of daughter cells failed to receive LDs from mother cells, in contrast to 14% in the daughter cells of the wild-type strain. In addition, a number of mutant strains exhibited diverse abnormalities in LD morphology. LDs with ring-like morphology were observed in spo7Δ and nem1Δ mutants, while enlarged LDs were seen in tgl3Δ. Several mutants, including loa1Δ, arv1Δ, srx1Δ, cla4Δ, cnm67Δ, sla2Δ, sto1Δ, and chc1Δ, showed increases in LD number. These findings provide important insights into the genetic regulation of lipid droplet behavior.

In yeast, Sey1p is the primary mediator of homotypic ER membrane fusion, whereas Dnm1p is well known for its role in mitochondrial and peroxisomal fission. However, the potential interplay between these machineries in ER network maintenance remains unclear. Here, I investigated the relationship between Dnm1p and Sey1p in Saccharomyces cerevisiae. Co-immunoprecipitation experiments demonstrated a physical association between Dnm1p and Sey1p, suggesting a possible connection between ER fusion and fission processes. Functional assays revealed that deletion of DNM1 resulted in inconsistent effects on in vitro ER microsome fusion activity, with both increased and decreased fusion observed in different experiments. Furthermore, in Sey1p-dependent proteoliposome fusion assays, the addition of Dnm1-MBP, as well as all control conditions—including boiled Dnm1-MBP, MBP alone, and TEV protease—led to a marked reduction in fusion compared to Sey1p alone, indicating non-specific inhibition rather than a direct regulatory effect of Dnm1p. These findings highlight the complexity of ER network regulation and indicate that while Dnm1p and Sey1p physically interact, Dnm1p does not play a considerable role in regulating Sey1p-mediated ER membrane fusion in vitro. To fully elucidate the functional significance of the Dnm1p-Sey1p interaction, future studies should employ optimized in vitro fusion assays and in vivo approaches to dissect the molecular mechanisms underlying ER dynamics.