Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast

TLDR

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a widely used tool for studying biological processes, and systematic studies of many genes would benefit from such assays. This study describes the use of GFP as a vital reporter molecule for localization and expression studies in Saccharomyces cerevisiae. The authors constructed GFP expression vectors enabling N‑ or C‑terminal fusion to genes of interest, used a gene‑replacement cassette to place gfp downstream of promoters, and quantified promoter activity in living yeast cells by flow cytometry while allowing simultaneous gene deletion. Analysis of three unknown ORFs revealed distinct staining patterns that predicted their subcellular localization, and the experiments demonstrate that GFP is a convenient fluorescent marker for both localization and gene expression studies in budding yeast.

Abstract

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted much attention as a tool to study a number of biological processes. This study describes the use of GFP as a vital reporter molecule for localization and expression studies in Saccharomyces cerevisiae. Construction of GFP expression vectors which allow N- or C-terminal fusion of the gfp gene to a gene of interest allowed the generation of fusion proteins whose subcellular localization was followed by fluorescence microscopy in living yeast cells. Analysis of three unknown open reading frames obtained from the budding yeast chromosome XIV resulted in distinct staining patterns, allowing prediction of the cellular localization of these unknown proteins. Furthermore, GFP was used to construct a gene replacement cassette which, after homologous integration into the genomic locus, placed the gfp gene behind a promoter of interest. The amount of GFP produced from this promoter was then quantified in living yeast cells by flow cytometry. With this novel replacement cassette a gene of interest can be deleted and at the same time its expression level studied under various growth conditions. The experiments presented here suggest that GFP represents a convenient fluorescent marker for localization studies as well as gene expression studies in budding yeast. Systematic studies of a large number of genes should benefit from such assays.