A Flow Cytometry-Based Phenotypic Screen To Identify Novel Endocytic Factors in Saccharomyces cerevisiae

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Endocytosis is crucial for cells to internalize plasma membrane components.
• Clathrin-mediated endocytosis (CME) is the primary internalization pathway, involving over 60 proteins.
• Novel genes regulating endocytosis and cargo selection remain to be identified.

Purpose of the Study:

• To identify novel genes involved in endocytosis using a high-throughput genetic screen.
• To characterize the roles of identified genes in cargo selection and endocytic machinery dynamics.
• To discover new components of the endocytic pathway in yeast.

Main Methods:

• Mutagenic screen in Saccharomyces cerevisiae using Mup1-pHluorin (pHl) cargo.
• Fluorescence-activated cell sorting to isolate mutants with impaired cargo trafficking.
• Subcellular localization analysis and whole-genome sequencing to identify and characterize mutations.

Main Results:

• Identified mutations in known endocytic genes (SLA2, SLA1, EDE1, ARP3) and novel genes (KRE33, IQG1).
• Characterized defects in endocytic machinery dynamics and actin nucleation in mutant strains.
• Validated the screening approach for discovering new endocytic pathway components.

Conclusions:

• The study successfully identified novel genes, KRE33 and IQG1, implicated in endocytosis.
• The findings expand our understanding of the protein network regulating CME.
• The developed screening method is effective for discovering new endocytic genes.