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

Abstract

The fusion of fluorescent proteins (FPs) to endogenous proteins is a widespread approach for microscopic examination of protein function, expression, and localization in the cell. However, proteins that are sensitive to FP fusion or expressed at low levels are difficult to monitor using this approach. Here, we develop a single-chain fragment variable (scFv)-FP approach to efficiently label Saccharomyces cerevisiae proteins that are tagged with repeats of hemagglutinin (HA)-tag sequences. We demonstrate the successful labeling of DNA-binding proteins and proteins localized to different cellular organelles including the nuclear membrane, peroxisome, Golgi apparatus, and mitochondria. This approach can lead to a significant increase in fluorescence intensity of the labeled protein, allows C'-terminal labeling of difficult-to-tag proteins and increased detection sensitivity of DNA-damage foci. Overall, the development of a scFv-FP labeling approach in yeast provides a general and simple tool for the function and localization analysis of the yeast proteome.