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J. Cell Biol.,
Volume 142, Number 4, August 24, 1998 949-961
Departments of Surgery and Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201
The yeast a-factor receptor (encoded by
STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis.
1996. J. Cell Biol. 134:661-674) and both depend on sequence elements within the receptor's regulatory, cytoplasmically disposed, COOH-terminal domain (CTD).
Here, we concentrate on the Ste3p sequences required
for constitutive endocytosis. Constitutive endocytosis is
rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole
where it is degraded, all with a t1/2 of 15 min. Deletion
analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD
that is the minimal sequence required for this rapid
turnover. Deletions intruding into this interval block or
severely slow the rate of endocytic turnover. Moreover,
the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for
ubiquitination and endocytosis, this sequence also is
sufficient. When transplanted to a stable cell surface
protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis
and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval
highlights its overall complexity
no singular sequence
motif or signal is found, instead required sequence elements distribute throughout the entire interval. The
high proportion of acidic and hydroxylated amino acid
residues in this interval suggests a similarity to PEST
sequencesa broad class of sequences which have
been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and
cytoplasmic proteins. A likely possibility, therefore, is
that this sequence, responsible for both endocytosis and
ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that
the true signal in the wild-type receptor extends beyond
the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which
extend another 21 residues to the COOH terminus of
Ste3p. Together with sequences identified in two other
yeast plasma membrane proteins, the STE3 sequence
defines a new class of ubiquitination/endocytosis signal.
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