Résumé
This chapter describes the yeast endocytosis assays. Lucifer Yellow carbohydrazide (LY) is asmall fluorescent molecule that can be used as a marker of fluid-phase endocytosis in mammalian systems. Internalized LY accumulates in the yeast vacuole and can easily be visualized by fluorescence microscopy. Results obtained with LY are best interpreted when combined with the results from α-factor uptake and degradation assays. The only specific marker of endocytosis by yeast cells described to date is the pheromone α-factor, α-Factor binds to the α-factor receptor on a cells. Using α-factor one can distinguish three distinct events: binding to cell surface receptors, internalization of the pheromone, and degradation of internalized α-factor. Recently, researchers have detected a vesicular intermediate in the transport of α-factor to the vacuole. The α-factor carded in this vesicle is protected from protease digestion by the membrane and floats in density gradients. The identification of this vesicular intermediate provides evidence for the delivery of α-factor to the vacuole via an endocytic mechanism. α-factor internalization is to date the most reliable way to quantitatively assess the earlier stages of endocytosis in yeast. An inability to degrade internalized α-factor could reflect, depending on the circumstances, the absence of vacuolar hydrolase activity or a defect in transport of α-factor to the vacuole.