Techniques update: A genetics-friendly GFP assay

Abstract

Green fluorescent protein (GFP) has become a popular reporter protein for research in cell biology. When used in yeast, GFP expression is usually monitored by fluorescence microscopy or flow cytometry1xGardner, R.G. et al. Mol. Biol. Cell. 1998; 9: 2611–2626Crossref | PubMedSee all References. However, the lack of a colony-level assay for GFP expression has somewhat limited the genetic applications of this powerful tool.We have developed a straightforward technique for direct examination of GFP fluorescence in living yeast colonies on solid medium. To examine colony fluorescence, agar plates must be illuminated with intense light of the appropriate wavelength, and the emitted fluorescence must be detected separately from the very intense background of reflected excitation light. For the S65T version of GFP, the excitation wavelength is 488 nm. A standard slide projector (Kodak Carousel™ 4400) provides luxuriant illumination when fitted with a 488 nm, narrow-bandpass filter. Such filters can be custom-made by Omega Optical (www.omegafilters.com). The 488 nm field produced by the filtered projector allows simultaneous examination of multiple agar plates. The green fluorescence emitted from the colonies is visualized by viewing the illuminated plates through a long-bandpass cutoff filter that only blocks passage of the excitation light. For this, we use a Kodak Wratten No. 12 filter, available in any camera store, which can be taped to a clear face mask, goggles or glasses.We use the GFP reporter to study the regulated degradation of the essential enzyme HMG-CoA reductase (HMGR). This protein is an integral-membrane resident of the endoplasmic reticulum that undergoes degradation as part of cellular control of sterol synthesis. One of the ways in which we study the degradation of the yeast HMGR isozyme Hmg2p is to use a fusion protein of Hmg2p and GFP that undergoes physiologically regulated degradation and is detectable by a variety of optical techniques2xSee all References.The degradation rate of the Hmg2p–GFP determines the in vivo steady-state levels of the protein, and hence, the cellular fluorescence. We are using the GFP colony assay to screen for mutant yeast strains with alterations in steady-state level. For example, in hrd1-1 mutants, Hmg2p–GFP degradation is blocked, and the in vivo levels of the reporter are higher than in a strain with a wild-type (wt) HRD1 gene3xHampton, R.Y., Gardner, R.G., and Rine, J. Mol. Biol. Cell. 1996; 7: 2029–2044Crossref | PubMedSee all References. This difference in cellular fluorescence is readily apparent at the colony level when viewed with the projector illuminator. Fig. 1Fig. 1 shows patches of otherwise identical yeast strains expressing the Hmg2p–GFP, in which degradation is normal (wt) or deficient (hrd1-1). Although the colonies look identical by direct illumination (Fig. 1bFig. 1b), the difference in fluorescence is readily apparent when examined for fluorescence (Fig. 1aFig. 1a).Figure 1(a) Green fluorescent protein (GFP) fluorescence in wild-type (wt) and hrd1-1 cells. (b) Same field as (a) but viewed by direct illumination. (Reproduced, with permission, from Ref. 2xSee all References2.)View Large Image | View Hi-Res Image | Download PowerPoint SlideWe are using the colony-fluorescence assay in a variety of genetic approaches to identify colonies with greater or lesser levels of Hmg2p–GFP. It is important to emphasize that the approach clearly depends on the levels and distribution of the GFP reporter being studied. However, we believe that this simple technique will have many applications in yeast as well as in other organisms that are amenable to GFP studies.