Chapter 8: GFP Fusion Proteins as Probes for Cytology in Fission Yeast

Abstract

This chapter discusses many practical considerations for expressing green fluorescent protein (GFP) fusion proteins in fission yeast, with an emphasis on guidelines and examples rather than on specific rules or recipes. There are primarily two areas in which GFP fusion proteins may be particularly useful in fission yeast. First, new types of experiments, such as determining protein localization in cells without the potential artifacts of fixation or filming protein localization and dynamics in living yeast cells, have now become possible. In addition, using GFP fusion proteins rather than more conventional methods for seeing proteins inside cells may make specific techniques, such as visual screens for protein localization, much more simple and rapid in execution. Expressed GFP fusion proteins generally need to be visible to the naked eye under the microscope, although if sensitive imaging equipment is available, the expression only just at the limit of detection by the eye may be sufficient. Producing a visible protein may often require higher than normal expression levels and can be achieved by using heterologous promoters, multicopy plasmids, or both. In addition, GFP mutants can now be used in place of the wild-type GFP.