Protein fluorescent labeling in live yeast cells using scFv-based probes

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Fluorescent protein (FP) fusion is common for studying protein function, expression, and localization.
• Proteins sensitive to FP fusion or expressed at low levels pose challenges for microscopic analysis.
• Existing methods struggle with accurate visualization of certain yeast proteins.

Purpose of the Study:

• To develop an efficient labeling method for difficult-to-tag Saccharomyces cerevisiae proteins.
• To enhance fluorescence intensity and detection sensitivity for low-abundance or sensitive proteins.
• To provide a versatile tool for analyzing yeast proteome function and localization.

Main Methods:

• Developed a single-chain fragment variable (scFv)-FP approach.
• Utilized hemagglutinin (HA)-tag sequences for protein tagging in yeast.
• Applied the scFv-FP system to label various proteins, including DNA-binding proteins and organelle-localized proteins.

Main Results:

• Successfully labeled DNA-binding proteins and proteins in diverse organelles (nuclear membrane, peroxisome, Golgi, mitochondria).
• Achieved significant increase in fluorescence intensity for labeled proteins.
• Demonstrated enhanced detection sensitivity for DNA-damage foci.
• Enabled C'-terminal labeling of challenging proteins.

Conclusions:

• The scFv-FP approach offers an efficient and generalizable tool for yeast protein analysis.
• This method improves the study of protein function and localization in Saccharomyces cerevisiae.
• The technique increases detection sensitivity, aiding research on DNA-damage and other cellular processes.