THE SEQUENCE NPFXD DEFINES A NEW CLASS OF ENDOCYTOSIS SIGNAL IN SACCHAROMYCES-CEREVISIAE

The yeast membrane protein Kex2p uses a tyrosine-containing motif with in the cytoplasmic domain for localization to a late Golgi compartment . Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi locali zation sequence or other sequences in the Kex2p cytoplasmic domain med iate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor, Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, und ergoes rapid endocytosis that is dependent on clathrin and End3p. Upta ke of Stex22p does not require the Kex2p Golgi localization motif. Ins tead, the sequence NPFSD, located 37 amino acids from the COOH terminu s, is essential for Stex22p endocytosis, Internalization was abolished when the N, P, or F residues were converted to alanine and severely i mpaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking l ysine-based endocytosis signals present in wild-type Ste2p, An NPF seq uence is present in the cytoplasmic domain of the a-factor receptor, S te3p. Mutation of this sequence prevented pheromone-stimulated endocyt osis of a truncated form of Ste3p. Our results identify NPFSD as a cla thrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiqu itin-dependent endocytosis signals in yeast.