A LARGE PEST-LIKE SEQUENCE DIRECTS THE UBIQUITINATION, ENDOCYTOSIS, AND VACUOLAR DEGRADATION OF THE YEAST A-FACTOR RECEPTOR

The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constituti ve, ligand-independent mode. Both modes are associated with receptor u biquitination (Roth, A.F., and N.G. Davis. 1996, J. Cell Biol. 134:661 -674) and both depend on sequence elements within the receptor's regul atory, cyto plasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocyto sis. Constitutive endocytosis is rapid. Receptor is synthesized, deliv ered to the cell surface, endocytosed, and then delivered to the vacuo le where it is degraded, all with a t(1/2) of 15 min. Deletion analysi s has defined a 36-residue-long sequence mapping near the COOH-termina l end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severe ly slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this seq uence show undetectable levels of ubiquitination, and mutants having i ntermediate endocytosis defects show a correlated reduced level of ubi quitination. Not only necessary for ubiquitination and endocytosis, th is sequence also is sufficient. When transplanted to a stable cell sur face protein,the plasma membrane ATPase Pma1p, the 36-residue STE3 sig nal directs both ubiquitination of the PMA1-STE3 fusion protein as wel l as its endocytosis and consequent vacuolar degradation. Alanine scan ning mutagenesis across the 36-residue-long interval highlights its ov erall complexity-no singular sequence motif or signal is found, instea d required sequence elements distribute throughout the entire interval . The high proportion of acidic and hydroxylated amino acid residues i n this interval suggests a similarity to PEST sequences-a broad class of sequences which have been shown to direct the ubiquitination and su bsequent proteosomal degradation of short-lived nuclear and cytoplasmi c proteins. A likely possibility, therefore, is that this sequence, re sponsible for both endocytosis and ubiquitination, may be first and fo remost a ubiquitination signal. Finally, we present evidence suggestin g that the true signal in the wild-type receptor extends beyond the 36 -residue-long sequence defined as a minimal signal to include contiguo us PEST-like sequences which extend another 21 residues to the COOH te rminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ub iquitination/endocytosis signal.