Visualization of receptor-mediated endocytosis in yeast

We studied the ligand-induced endocytosis of the yeast alpha-factor recepto r Ste2p by immune-electron microscopy. We observed and quantitated time-dep endent loss of Ste2p from the plasma membrane of cells exposed to alpha-fac tor. This ligand-induced internalization of Ste2p was blocked in the well-c haracterized endocytosis-deficient mutant sac6 Delta. We provide evidence t hat implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the fin ger-like plasma membrane invaginations within actin cortical patches. Consi stent with this, we show that Ste2p is not located within the cortical acti n patch before and during receptor-mediated endocytosis. In wild-type cells exposed to alpha-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immune-electron micr oscopy experiments these compartments labeled with antibodies directed agai nst the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carb oxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeli ng technique we have colocalized antibodies against Ste2p and carboxypeptid ase Y to this compartment, thereby identifying these compartments as prevac uolar late endosomes.