We have characterized the sequences within the yeast a-factor receptor that direct its rapid, ligand-independent endocytosis. The essential sequence is striking, both in terms of its large size, minimally defined as 36 residues long, and its high density of both acidic amino acids and hydroxylated amino acids, particularly serine. Not only does this sequence specify uptake, but also ubiquitination. Receptor mutants that lack this sequence are not ubiquitinated and mutants partially deleted for this interval show a reduced level of ubiquitination that correlates well with their residual capacity for endocytosis. This sequence also is a sufficient, self-contained, transportable unit. When transplanted into the normally stable cell surface protein Pma1p, the STE3 sequence directs both ubiquitination and endocytosis: the result being delivery of the PMA1-STE3 fusion protein to the vacuole for degradation.
Examination of the STE3 signal by visual inspection and by alanine-scanning mutagenesis reveals no obvious similarities to the short L- or Y-based peptidyl signals that mediate clathrin-dependent uptake in mammalian cells (Mellman, 1996
; Schmid, 1997
). Furthermore, no example of the two sequences that have been previously associated with the ligand-dependent uptake modes of the two yeast pheromone receptors are apparent (Rohrer et al., 1993
; Tan et al., 1996
). The large size of the STE3 ubiquitination/endocytosis signal could reflect a folding requirement to a particular three-dimensional shape for functionality. Alternatively, the functional signal could consist of an extended string or cluster of certain classes of residues. In this regard, the high proportion of both acidic and hydroxylated residues within both the 36-residue minimal signal and the more inclusive 57-residue sequence is striking. These are two of the three key sequence elements of PEST sequences, a set of sequences that act as signals for ubiquitination and consequent proteosomal turnover for various short-lived cytoplasmic and nuclear proteins. The third element of PEST sequences are prolines; there are two prolines within the COOH-terminal 57 residues of Ste3p, however, neither locate within the 414–449 interval.
PEST sequences are responsible for directing the ubiquitin-dependent proteosomal degradation of a variety of short-lived cytoplasmic and nuclear proteins (Rechsteiner and Rogers, 1996
). Though PEST sequences share no primary sequence identity to one another, they do share overall character—protein sequences, 10–50-residues-long containing unusually high densities of prolines, the acidic residues glutamate and aspartate, and the hydroxylated amino acids serine and threonine. The resemblance of the STE3 signal to PEST sequences is consistent with the initiating role that ubiquitin has been proposed to play in Ste3p endocytosis (Roth and Davis, 1996
). The STE3 signal may be first and foremost a signal for ubiquitination.
Recent work on two other yeast plasma membrane proteins, the a
-factor export protein Ste6p and the uracil permease Fur4p, indicate that sequences similar to the STE3 signal likely participate in the uptake of these proteins as well (Kolling and Losko, 1997
; Marchal et al., 1998
). Like Ste3p, endocytosis and turnover of Ste6p and Fur4p also appear to be ubiquitin dependent (Kolling and Hollenberg, 1994
; Galan et al., 1996
; Loaza and Michaelis, 1998
). For Ste6p, deletion of an acidic 61-residue sequence rich in serines and threonines, abolished both Ste6p ubiquitination and its rapid turnover. Though this 61-residue-long STE6
sequence was not sufficient to direct endocytosis of a PMA1-STE6 fusion construct (equivalent in design to the PMA1-STE3 constructs used herein), a larger, more inclusive 108-residue sequence did suffice (Kolling and Losko, 1997
). Mutagenic studies on Fur4p, also demonstrate the involvement of a sequence rich in acidic and hydroxylated residues in uptake and vacuolar degradation of this permease (Marchal et al., 1998
). These two sequences, together with the STE3
sequence appear to constitute a new class of endocytosis signal—a class where the primary function of the signal may be to instigate ubiquitination.
Though resembling PEST sequences, these three ubiquitination/endocytosis signals do not score strongly as PEST sequences. This algorithm (Rogers et al., 1986
) has proved to be a powerful predictor of proteins subject to rapid ubiquitin-dependent proteosomal turnover. Nonetheless, as there is little direct molecular data concerning the essential features of PEST sequences, the strictures of this algorithm are necessarily somewhat arbitrary. The algorithm examines only those sequences that are bounded by basic residues (K, R, or H), and that include at least one Pro, at least one acidic residue (D or E), and at least one hydroxylated amino acid (S or T). Of these, the strength of the resulting PEST score mostly depends on the proportion of amino acids within the interval that are either P, D, E, S, and T. The failure of the three ubiquitination/endocytosis signals to score strongly as PEST sequences reflects the paucity of proline residues within these sequences. This could indicate either that the PEST algorithm is overly stringent with regards to its proline requirement, or more interestingly, a point of functional divergence of these signals with PESTs. Although there appears to be significant overlap in the enzymatic machinery catalyzing the ubiquitination of both proteosomal substrates and cell surface proteins targeted for endocytosis (see below), the ubiquitination requirements for these two processes may be distinct. Where formation of a multi-ubiquitin chain on the substrate is generally required for proteosomal recognition (Hochstrasser, 1996
), recent work on the α-factor receptor indicates that the addition of a single ubiquitin moiety suffices to direct endocytosis (Terrell et al., 1998
). One possibility, therefore, is that the prolines present in the PEST sequences of proteosomal substrates play a role in specifying the construction of the multi-ubiquitin chain. In any case, as the relationship of these new endocytosis signals to PEST sequences remains uncertain, we perpetuate the term “PEST-like,” coined by Marchal et al. (1998)
in their description of the Fur4p signal.
The 36-residue-long STE3 signal is the minimal sequence sufficient for ubiquitination and endocytosis. PEST-like sequences in STE3, however, extend to the COOH terminus and include in addition to the 36 residues (414–449), the contiguous 21 COOH-terminal residues (450–470). We have presented evidence that this COOH-terminal sequence (450–470) may participate together in redundant fashion with the 414–449 signal. Similar redundancy may also apply within the minimal 414–449 interval. Loss of function was gradual with extension of the deletions into this interval. With extension of the deletion from residue 450 to residue 447, the t1/2 increased from 25 to 40 min (Fig. , compare Δ450–468 and Δ447–468). With further extension to residue 441 or to residue 434, the t1/2 increased to 70 min (Fig. , Δ441–468 and Δ434–468). Only deletions extending to residue 423 and residue 413 effectively abolished endocytosis (Fig. , Δ423–468 and Δ413– 468). Thus it appears that both the rate of endocytosis, and also the degree of ubiquitin modification (Fig. ) reflects the length of the PEST-like domain remaining in each mutant. Functionality of the signal perhaps depends on its overall size and on overall density of acidic and hydroxylated residues. Larger, more negatively charged sequences may provide better substrates for ubiquitination.
Negative charge within a potential ubiquitination/endocytosis signal could be further increased through the introduction of phosphates. Such a model has been suggested both for Fur4p endocytosis (Marchal et al., 1998
) and for the ligand-dependent endocytosis of Ste2p (Hicke and Riezman, 1996
; Hicke et al., 1998
). Addition of acidic phosphate moieties within an already acidic domain perhaps elevates a sequence through some threshold barrier of required negative charge density, allowing recognition by the E3 ubiquitination machinery. For a number of diverse proteosome substrates, phosphorylation within PEST sequences often precedes and serves to instigate subsequent ubiquitination (Hochstrasser, 1996
; Rechsteiner and Rogers, 1996
). This may be true for Ste3p as well, as it clearly is subject to phosphorylation (Roth and Davis, 1996
In terms of potential ubiquitin acceptor sites, Ste3p has a total 23 cytoplasmically available lysine residues. Three map within the COOH-terminal 57-residue PEST-like sequence and of these, two within the 36-residue minimal signal. The disabling effect of the triple alanine mutation of residues 432–434 (Fig. ) suggested the possibility that Lys432 might serve as the sole ubiquitin acceptor site within the Δ450–468 receptor. However, this is not supported by the effects of a K432R point mutation. Within the context of the Δ450–468 receptor, this mutation failed to block receptor ubiquitination and showed only an intermediate turnover disability (t1/2 = 60 min; data not shown). Possibilities currently being tested are either that the ubiquitination occurs at lysines mapping outside of the PEST-like sequence and/or that multiple lysines are used redundantly.
The endocytosis signal characterized herein is also a ubiquitination signal. While decoding this signal could involve the binding of the adaptins and coat proteins, it seems more likely that initial interactions are with enzymes that catalyze ubiquitin addition. Generally for ubiquitination, although the precise biochemical or genetic requirements are incompletely understood, it is clear that the E2 and E3 classes of enzymes participate both in recognizing the substrate protein and in the subsequent ubiquitin transfer (Hochstrasser, 1996
). For Ste3p ubiquitination, there are several E2 and E3 candidates. A component of the E3 complex could be the hect-domain protein Rsp5p. Though involvement in pheromone receptor endocytosis remains to be determined, RSP5
is required for the ubiquitination and endocytic turnover of several different yeast plasma membrane proteins (Hein et al., 1995
; Lucero and Lagunas, 1997
). In terms of participating E2 functions, the redundant enzymes Ubc4p and Ubc5p, as well as Ubc1p are likely involved as they have been implicated in the ubiquitination and endocytosis for a variety yeast plasma membrane proteins (Kolling and Hollenberg, 1994
; Egner and Kuchler, 1996
; Hicke and Riezman, 1996
; Roth and Davis, 1996
). These E2 and E3 activities, however, can not be exclusively devoted to plasma membrane protein turnover. Rsp5p, Ubc4p, and Ubc5p also are required for the ubiquitination and proteosomal turnover of a variety of short-lived cytoplasmic and nuclear proteins (Hochstrasser, 1996
). In addition, Rsp5p recently has been implicated as a participant in the mitochondrial import process (Zolladek et al., 1997
). Thus, these enzymes participate in a number of quite distinct cellular processes occurring at a number of distinct cellular sites. Specificity for membrane proteins will likely also involve the inclusion of other, presently unknown factors into these E2 and E3 complexes to direct both cellular localization and proper substrate recognition. A goal for the future, then, is to identify the specific factors that bind to and decode this new class of ubiquitination/endocytosis signal.