Large-Scale Analysis of Membrane Transport in Yeast Using Invertase Reporters
Transport of membrane proteins between cellular organelles requires the concerted action of many regulatory factors, which aid in cargo recognition and vesicle formation, targeting, and fusion. The yeast Saccharomyces cerevisiae is a useful model system for studying such regulators, due to the availability of genome-wide mutant collections and reporter proteins that provide sensitive biochemical readouts of individual transport pathways. Here, we describe an enzymatic invertase assay for evaluating endocytic recycling using a chimeric GFP-Snc1-Suc2 reporter. Cell surface levels of this reporter can be measured by a colorimetric assay that monitors sucrose hydrolysis at the plasma membrane, using two different methods. The first is a semiquantitative agar overlay assay followed by image densitometry that is suitable for high-throughput screening of arrayed yeast colonies. In the second, more quantitative assay, an enzymatic solution is added to yeast cultures in a multi-well plate and the absorbance is assessed by a plate reader. Furthermore, the modular nature of the chimeric reporter allows alternate transport signals to be introduced, thereby expanding the range of transport pathways that can be evaluated by this method. Together these techniques can be used to explore the function of genes involved in a variety of cellular trafficking pathways.
Key wordsYeast Deletion mutant collection Phenotypic screening Vesicle transport Golgi Endosome Endocytosis Snc1 GSS Invertase
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