Departments of Surgery and Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201
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Abstract |
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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 complexityno 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
sequences
a 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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Introduction |
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IN mammalian cells, the events surrounding the initial
cell surface capture of membrane proteins by receptor-mediated endocytosis are reasonably well understood (Mellman, 1996; Schmid, 1997
). The process is selective with proteins destined for uptake being first
sequestered into differentiated subdomains of the plasma membrane, i.e., clathrin-coated pits. Pits invaginate, pinching off as clathrin-coated membrane vesicles into the cytoplasm. Uptake selectivity is controlled through a specific
binding interaction between the endocytosis signal, a peptidyl sequence localized to the cytoplasmically exposed
domains of the membrane protein, and the adaptin complex, a component of the coated pit. Two varieties of such
endocytosis signals have so far been identified. Y-based signals constitute a loosely conserved motif in which an aromatic residue, either tyrosine or phenylalanine plays a
central role. L-based signals have a leucine-leucine dipeptide as the central recognizable feature. For both, the functional signal is short and discrete, lying within a polypeptide sequence 5-10 residues long.
In the yeast Saccharomyces cerevisiae, much progress has
been made in recent years in understanding endocytic processes. Genetic analyses have identified a large collection of
cellular proteins that participate in this process, both at the
initial plasma membrane uptake step as well as at the subsequent endosomal transport steps (Riezman et al., 1996). In
spite of the wealth of genetic data, the molecular details of
the initial cell surface uptake event remain surprisingly obscure. As for mammalian cells, the process is selective
only a subset of the plasma membrane proteins are subject
to endocytosis, others, for instance, the plasma membrane ATPase, Pma1p, remain stably at the cell surface (Benito et
al., 1991
). Although clear homologues of the mammalian
clathrin and adaptins have been identified, their direct involvement in this uptake remains uncertain. Mutants generated in the gene for the yeast clathrin heavy chain slow, yet
do not block endocytic uptake (Payne et al., 1988
; Tan et al.,
1993
). Much more profound effects are seen instead for mutants that disrupt the yeast cell's actin cytoskeleton. Mutants in actin, myosin, as well as in a variety of actin-associated proteins often lead to a near total block of the uptake
process (Riezman et al., 1996
).
Protein ubiquitination has also emerged as a regulator
of initial endocytic uptake events both in yeast (Kolling
and Hollenberg, 1994; Egner and Kuchler, 1996
; Galan et al.,
1996
; Hicke and Riezman, 1996
; Roth and Davis, 1996
;
Galan and Haguenauer-Tsapis, 1997
; Kolling and Losko,
1997
; Marchal et al., 1998
) and in mammalian cells (Strous
et al., 1996
; Gowers et al., 1997
). Ubiquitin, a small 76-residue protein, has long been known as a regulator of protein degradation in the cell cytoplasm. Covalent attachment of ubiquitin to selected lysyl residues targets the
substrate protein for proteosomal degradation (Hochstrasser, 1996
). The degradative turnover of plasma membrane proteins or of proteins that enter the cell via the endocytic route has generally been thought to involve a
separate system, that being proteolysis by the proteases residing within the membrane-enclosed space of the lysosome. Nevertheless, for a number of years, a growing collection of mammalian cell surface proteins have found to
have covalently attached ubiquitin (Ciechanover, 1994
).
Recently, these findings have been mirrored by similar
findings in yeast where a number of different plasma
membrane proteins have been found to be ubiquitinated. In yeast, studies designed to test the function of the ubiquitin modification draw a consistent picture, linking ubiquitination to endocytosis (Kolling and Hollenberg, 1994
;
Egner and Kuchler, 1996
; Galan et al., 1996
; Hicke and
Riezman, 1996
; Roth and Davis, 1996
; Kolling and Losko,
1997
; Galan and Haguenauer-Tsapis, 1997
; Marchal et al.,
1998
). The general model that has emerged has ubiquitin
attached to cytoplasmic portions of surface membrane proteins serving as a flag, signaling uptake. Strong support
for this model comes from work on the
-factor receptor,
one of the two pheromone receptors in yeast. In a first report, mutation of the presumptive lysyl ubiquitin acceptor
site concomitantly abolished both ubiquitination and uptake (Hicke and Riezman, 1996
) indicating a requirement
for ubiquitination in endocytosis. In a second report, ubiquitin addition was found to provide a sufficient trigger for
uptake: a translational in-frame fusion of a ubiquitin moiety to the cytoplasmic domain of an endocytosis-defective
mutant
-factor receptor restored endocytic capacity (Terrell et al., 1998
). Furthermore, the results of Terrell et al.
(1998)
also indicate a fundamental point of difference between ubiquitin's involvement in proteosomal degradation
versus endocytosis. Whereas proteosomal recognition generally requires the elaboration of a multi-ubiquitin chain
on the substrate protein (Hochstrasser, 1996
), addition
of a single ubiquitin moiety appears sufficient to specify
-factor receptor endocytosis (Terrell et al., 1998
).
Both of the yeast pheromone receptors, the a- and -factor receptors, have been well studied as paradigms for endocytosis. These two G protein-coupled receptors mediate the hormonal communication that precedes the sexual
conjugation of the two yeast haploid mating types, the a
cell with the
cell. Though sharing no primary sequence
homology, both receptors couple to the same downstream
signaling pathway (Bender and Sprague, 1986
). Our analyses have focused mainly on the a-factor receptor encoded
by the gene STE3. Ste3p has the canonical G protein-coupled receptor structure with seven transmembrane domains followed at the COOH terminus by, in the case of
Ste3p, a relatively large 185-residue hydrophilic, cytoplasmically exposed, COOH-terminal tail domain (CTD).1
The CTD is a regulatory domain and can be largely deleted without impairing the gross functioning of the receptor, i.e., ligand binding and G protein coupling (Boone
et al., 1993
). Though functional, receptors truncated for
this CTD manifest an unregulated response to the a-factor
ligand that is both exaggerated and prolonged. In addition,
these mutant receptors are defective for the two uptake
modes that have been demonstrated for this receptor
a
constitutive or ligand-independent uptake mode, as well as
a ligand-dependent endocytosis (Davis et al., 1993
; Tan et al.,
1996
). Both modes involve ubiquitination (Roth and
Davis, 1996
) and both deliver surface receptor to the vacuole for degradation by the resident proteases (the vacuole
is the yeast equivalent organelle to the mammalian lysosome). Though both receptors are subject to the two uptake modes, for the a-factor receptor, constitutive endocytosis is by far the more prominent, transporting unliganded receptor from surface to vacuole for degradation with a t1/2
of 15 min. Furthermore, at least for Ste3p, the two uptake
modes are mechanistically distinct. The two are controlled
by distinct signals mapping to distinct portions of the receptor CTD (Davis et al., 1993
; Tan et al., 1996
). Second,
the two are distinguished by the participating cellular
functions required for each: mutants in the ankyrin-repeat
protein Akr1p block constitutive endocytosis of the a-factor receptor, yet have no effect on ligand-dependent uptake (Givans and Sprague, 1997
). In spite of these differences, much of the mechanism may also be shared.
Receptor ubiquitination and vacuole-directed transport
are common to both and both are blocked by mutants in
the actin-associated function SLA2/END4 (Roth and Davis,
1996
), suggesting a requirement of the actin cytoskeleton
for both uptake modes.
In the present work we characterize the sequences
within the a-factor receptor CTD required for its rapid
ligand-independent endocytosis, i.e., the constitutive endocytosis signal. Although an expanding collection of
plasma membrane proteins have now been shown to undergo endocytosis in yeast, the signals that direct uptake
have been studied in just a few cases. For the two pheromone receptors, sequences required for ligand-dependent
endocytosis have been characterized. The -factor receptor CTD sequence DAKSS has been shown to be required
for ligand-dependent uptake of a mutant receptor deleted
for the COOH-terminal two-thirds of its 128-residue-long
CTD (Rohrer et al., 1993
). The central lysine in this sequence appears to serve as the acceptor site for ubiquitin attachment (Hicke and Riezman, 1996
). For the a-factor receptor, the sequence NPFXD is required for ligand-dependent endocytosis (Tan et al., 1996
).
The work that follows characterizes the sequences
within the a-factor receptor, which direct its rapid, constitutive endocytosis. We find that a 36-residue-long sequence directs both uptake as well as the associated ubiquitination of the receptor. No obvious resemblance to
either the mammalian L- or Y-based endocytosis signals
or to the two signals characterized for the ligand-dependent uptake of the two yeast pheromone receptors is apparent. Instead, rich in both acidic and hydroxylated
amino acids, the Ste3p signal bears a resemblance to the
PEST sequences that direct ubiquitination and proteosomal turnover of short-lived cytoplasmic and nuclear proteins. Recent reports on two other yeast plasma membrane proteins, the a-factor export protein Ste6p and the
uracil permease Fur4p, identify similar PEST-like sequences as participating in their constitutive endocytosis
(Kolling and Losko, 1997; Marchal et al., 1998
). Together
these three sequences likely represent a new class of endocytosis signal
signals where the primary function may
be to direct the addition of an initiating ubiquitin.
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Materials and Methods |
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Plasmids
Three URA3/2µ plasmids were constructed for CUP1-driven overexpression of wild-type or myc-tagged ubiquitin (Ellison and Hochstrasser, 1991). pND186 (CUP1-ubiquitin), pND747 (CUP1-myc-ubiquitin), and pND187 (CUP1 promoter only) were constructed through the replacement of the ClaI-BamHI interval of YEp24 with the CUP1-ubiquitin-
containing ClaI-BamHI fragment from the corresponding URA3/CEN/
ARS plasmids pND164, pND165, and pND167, respectively (Roth and
Davis, 1996
).
The other plasmids constructed and used in this work divide into series of equivalent constructs carrying either wild-type STE3 or STE3 mutant versions. The strategy used in the construction of each series is reported in the following three sections.
Construction of In-Frame Deletions
For constructing in-frame deletions within the Ste3p CTD, the general strategy involved introduction of XhoI restriction sites by oligonucleotide-directed mutagenesis at various positions within the STE3 CTD-encoding sequences. Restriction and then ligation of upstream to downstream sites deletes the interval in between. Each XhoI site mutation replaced two adjacent STE3 codons with the sequence CTCGAG. Each replacement encoded the dipeptide leucine-glutamate and ligation of any two sites results in in-frame translation across the deletion.
Eight XhoI site mutations were constructed, at each of the following dipeptide codons: Leu398Lys399, Phe413Asp414, Ser423Lys424, Leu433His434, Phe441Glu442, Leu446Cys447, Pro450Ala451, and Ser458Ser459. In addition, a SalI site compatible with the XhoI sites was introduced at the Leu320Leu321 dicodon changing it to Val320Asp321.
Oligonucleotide-directed mutagenesis was by the method of Kunkel et al.
(1987). The ssDNA template was derived from pSL1839, a 5.5-kb
BamHI-SalI fragment from the original STE3 genomic library isolate
(Hagen et al., 1986
) carried on pRS306 (Sikorski and Hieter, 1989
). Mutant plasmids for each restriction site were subjected to DNA sequencing
in the vicinity of the site to confirm the fidelity of the mutagenesis.
Two additional SalI restriction sites, compatible with the XhoI site
reading frames, were also used: the natural SalI site at STE3 codons 364 and 365, as well as a SalI site introduced via linker ligation to the PstI site
located at STE3 codons 466-468 (see pSL1922 in Davis et al., 1993). This
linker-derived SalI site was used in combination with the different XhoI
site mutants to construct the series of "COOH-terminally"-truncated
STE3 mutants. In fact, these truly are in-frame deletions, as they all retain the natural COOH-terminal STE3 dipeptide Gly469Pro470.
Assessment of receptor ubiquitination levels required that each of the
receptor mutants be overexpressed from the GAL1 promoter. The plasmid pSL552 (Bender and Sprague, 1986) has a GAL1-controlled wild-type
STE3 gene carried on the yeast URA3/CEN/ARS plasmid YCp50 (Rose
et al., 1987
). In-frame STE3 deletions constructed above in the pSL1839
plasmid context, were introduced into pSL552 via replacement of 380-bp
SalI-SacI restriction fragment covering the COOH-terminal coding portion of STE3, with the equivalent fragment containing the desired deletion mutation.
Construction of Triple Alanine Substitution Mutations
The 12 triple alanine mutations created within the 414-449 interval of
STE3 (see Fig. 8) also were constructed via oligonucleotide-directed mutagenesis (Kunkel et al., 1987). For each, three adjacent STE3 codons are
replaced by the sequence GCTGCAGCC, encoding three consecutive alanines. This substitution also introduces a PstI site, allowing identification
of the mutant clone by restriction analysis. The ssDNA template for mutagenesis was from pND210, the
450-468 mutant version of pSL1839. Fidelity of mutagenesis was confirmed for each via DNA sequencing in the
vicinity of mutation site.
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Two of the triple alanine mutations were also constructed within the
context of wild-type (i.e., full-length) STE3. Reconstruction of STE3 with
either the 417-419 or 426-428 triple alanine mutations (see Fig. 9) used
the HindIII site at codons 433, 434, allowing the wild-type COOH-terminal STE3 sequences to be restored to the STE3450-468 versions of these
mutations.
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Construction of PMA1-STE3 Fusions
The plasmid pXZ28 (Harris et al., 1994) has a GAL1-driven HA epitope-
tagged PMA1 gene carried on a yeast URA3/CEN/ARS plasmid. The single HA epitope tag inserted near the NH2-terminal coding sequences, following the second codon of PMA1 does not affect PMA1 function. The
750-bp EcoRI-XhoI pXZ28 GAL1 promoter fragment plus the 3.6-kb
XhoI-PstI HA-PMA1-containing pXZ28 fragment were inserted into the
polylinker of the yeast LEU2/CEN/ARS vector pRS315 (Sikorski and Hieter, 1989
) between EcoRI and SacI to give pND533. This required modification of the PstI end of the 3.6-kb fragment by addition of a SacI linker.
The unique XhoI site of pND533, at the fusion boundary between GAL1
and PMA1 sequences was destroyed via end-filling and re-ligation to give
pND542. To create pND563, the immediate progenitor of PMA1-STE3
fusion protein plasmids, a new XhoI site was introduced into PMA1 by oligonucleotide-directed mutagenesis at COOH-terminal position two
residues from the end of the coding sequences (the XhoI site substitutes
for the codons Lys916Glu917). The PMA1 XhoI site is designed to accommodate in-frame fusions with the STE3 XhoI sites. For most of these fusions the XhoI site at STE3 codons 398, 399 was used (see above) with the
contributed STE3 sequences extending from codon 400 to different
COOH-terminal endpoints. For instance, to construct the PMA1-
STE3(400-440) fusion protein, the 398, 399 XhoI site was introduced via
oligonucleotide-directed mutagenesis into the STE3
441-468 deletion
mutant context. From this plasmid, a 250-bp XhoI-SacI fragment could be
excised (the SacI site is located 3' of the STE3 open reading frame [ORF])
and used to replace the XhoI-SacI fragment from the GAL1-HA-PMA1
plasmid pND563.
Strains
Genotype and source for some of the yeast strains used in this work are
reported in Table I. The two new strains listed, NDY343 and NDY344,
isogenic ste3::LEU2 versions of the strains NDY334 and NDY335 (Roth
and Davis, 1996
), were constructed via the two-step gene replacement
strategy (Boeke et al., 1987
) as described previously (Roth and Davis,
1996
), wherein a ste3
::LEU2 allele was used to replace STE3. The ste3
::
LEU2 allele, carried on the integrating plasmid pSL2165, a pSL1839 derivative (see "Plasmids"), in which the entire STE3 ORF is replaced by
the 2.85-kb BglII LEU2 fragment. The ste3 deletion extends from a point
417 bp upstream of the STE3 ORF to a point 111 bp downstream.
pSL2165 was uniquely restricted at a SfiI site located upstream of STE3 to
direct chromosome insertion.
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In addition to the strains reported in Table I, numerous other strains
were constructed and used in this work. The turnover of each Ste3p mutant
was measured in strains in which the mutant STE3 allele replaces the ste3::
LEU2 allele of NDY343. This strain construction used the two-step gene replacement strategy (Boeke et al., 1987
) as previously described (Roth and
Davis, 1996
). For each, the transplacing allele, derivatives of the integrating plasmid pSL1839 with the mutant STE3 in place of wild-type STE3, were
restricted with HpaI to direct chromosomal insertion. This eliminated one
potential source of experimental variability
differences in receptor expression levels resulting from clonal fluctuations in plasmid copy number.
Antibodies
Rabbit antisera was raised against the STE3 CTD, as part of one of two
bacterial fusion proteins, TrpE-STE3 (Clark et al., 1988) or MalE-STE3.
All Ste3p-specific antisera were affinity purified to the MalE-STE3 antigen coupled to CNBr-activated Sepharose 4B (Sigma Chemical Co., St.
Louis, MO). The HA.11 mAb (Berkeley Antibody Co., Berkeley, CA)
was used for detection of HA epitope-tagged PMA1-STE3 fusions. The
myc mAb (Oncogene Research, Uniondale, NY) was used for detection
of the myc 9E10 epitope-tagged ubiquitin (Ellison and Hochstrasser,
1991
). Pgk1p-specific antiserum was generously provided by T. Stevens
(University of Oregon, Eugene, OR).
Cell Labeling, Extract Preparation, and Immune Precipitation
Pulse-chase analysis (Roth and Davis, 1996) entailed in vivo labeling of
yeast cultures via a 10-min pulse of [35S]methionine, which was followed
by addition of the non-radioactive chase: methionine added to a concentration of 750 µg/ml, cysteine to 150 µg/ml, and yeast extract to 0.2%. At
various times after chase addition, culture aliquots were removed and extracts were prepared by the glass bead method described previously (Roth
and Davis, 1996
). Wild-type or mutant receptors were then isolated via
two iterative rounds of immunoprecipitation (Roth and Davis, 1996
).
Each round used different affinity-purified anti-Ste3p antibodies derived
from the polyclonal antisera of different rabbits. Immunoprecipitated
samples were subjected to SDS-PAGE, and then both to autoradiography
and to Storm PhosphorImaging (Molecular Dynamics, Inc., Sunnyvale,
CA) for the quantitative analysis.
For immunoprecipitation of the HA-tagged PMA1-STE3 fusion proteins, 20 µl of extract corresponding to 50 × 106 cells, prepared as described (Davis et al., 1993), was subjected to a single round of immunoprecipitation (Roth and Davis, 1996
) using 30 µl of a slurry of the HA.11
mAb conjugated to agarose beads (Berkeley Antibody Co.). Incubation
with the mAb in this case was for 16 h at 4°C.
Susceptibility to External Proteases
To assess the susceptibility of newly synthesized receptor to digestion by
extracellular proteases, cells were first subjected to in vivo labeling via the
pulse-chase protocol described above. At various times after chase addition, culture aliquots were removed and intact cells were treated with Pronase (Calbiochem-Novabiochem Corp., La Jolla, CA) and protein extracts were prepared (Davis et al., 1993). Immunoprecipitation and
further analysis was as described above.
Protein Extract Preparation, Western Analysis, and Phosphatase Treatment
For Western analysis, the relevant STE3 mutant or PMA1-STE3 fusion protein was expressed from the GAL1 promoter. For these experiments, overnight log-phase cultures in rich YP medium containing 2% raffinose were inoculated from minimal plates on which plasmid retention was selected. Induction of the GAL1 promoter involved the addition of galactose to 2%. The duration of the galactose induction was as indicated for each figure and was terminated when synthesis repression was desired with the addition of glucose to 3%. Protein extract preparation, SDS-PAGE, and Western analysis were as described previously (Roth and Davis, 1993).
Treatment of protein extracts with potato acid phosphatase (Boehringer Mannheim Corp., Indianapolis, IN) at a final concentration of 0.06 U/ml was as described previously (Roth and Davis, 1996).
Quantitation of the Proportion of Receptor Protein with Attached Ubiquitin
Quantitating the proportion of receptor proteins with attached ubiquitin
was by Western analysis of phosphatase-treated protein extracts for the
various STE3 mutants. Quantitative AMBIS densitometry of films (AMBIS Systems, Inc., San Diego, CA) resulting from ECL development of
the Western blot (Amersham Corp., Arlington Heights, IL) was used to
estimate the relative amount of the receptor protein present in the two
ubiquitin-modified electrophoretic positions (the presumptive mono- and
di-ubiquitinated forms; Roth and Davis, 1996), and at the unmodified position. Many aspects of this analysis rely on techniques wherein the measured output is produced non-linearly. To circumvent this potential source
of error, we have compared signals only of similar intensity levels. This
was achieved by applying the Western analysis to a dilution series for each
sample. For instance, with phosphatase-treated extracts derived from
wild-type Ste3p, the intensity level of the signal associated with the mono-ubiquitinated band was found to be intermediate between the level of the
unmodified receptor band present from the 1:5 and 1:25 dilutions of the
same extract. Quantitative densitometry of the band signals from the same
compared samples led to the determination that 10% of the wild-type receptor was present in the mono-ubiquitinated species band. As 10% was
also present in the di-ubiquitinated band, the summed estimate of 20%
was reported in Fig. 3 as proportion of receptor ubiquitinated.
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Results |
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Identification of a Receptor Subdomain That Directs Rapid Constitutive Endocytosis
Our previous work showed that the rapid constitutive endocytosis of Ste3p requires sequence elements within the
COOH-terminal 72-amino acid residues of the receptor
(Davis et al., 1993). To better delineate this requirement
we have constructed a series of in-frame deletion mutants
within the receptor's dispensable CTD. First we have first
investigated if CTD sequences in addition to the 72-residue COOH-terminal segment are required for rapid constitutive endocytosis. The 187-residue-long Ste3p CTD extends from Asp284 (the first charged residue after the
seventh transmembrane segment) to a COOH-terminal
Pro470. Three large deletion mutants, deleting residues 320-
363 (STE3
320-363), residues 365-414 (STE3
365-414), and the COOH-terminal residues 413-468 (STE3
413-468)
were used. As a first assessment of endocytosis, the rate of
degradative turnover of each of the different receptor mutants was determined (Fig. 1 A). While wild-type Ste3p
turns over rapidly with a t1/2 estimated to be ~15 min, the
413-468 mutant receptor is completely stable, indicating
that the COOH-terminal 58 residues are required for
this ligand-independent receptor turnover. Like wild-type, both the
365-414 and
320-363 receptors show rapid
turnover consistent with endocytosis that is largely unimpaired. Thus, of the STE3 CTD sequences, the COOH-terminal 58 residues play the primary role in specifying
rapid turnover. We have therefore concentrated our analyses on this interval.
The turnover defect of Ste3365-468p, a mutant missing
its COOH-terminal 105 residues, is a consequence of
blocked endocytic transport from surface to vacuole
(Davis et al., 1993
). With endocytosis blocked the receptor
accumulates at the cell surface. If the turnover defect of
the
413-468 mutant receptor also results from impaired endocytosis, then this receptor should also accumulate at
the cell surface. To assess surface localization of the
413-
468 receptor, we have used protease susceptibility as a
probe, testing the accessibility of the receptor within
whole cells to digestion by added, external protease. This
assay distinguishes surface-localized receptor (susceptible
to external proteases), from receptor that localizes to compartments inside the cell (resistant to external proteases)
(Davis et al., 1993
). As for Fig. 1 A, cells were subjected to
pulse-chase analysis. At various times after the initiation of the non-radioactive chase, aliquots of the labeled, intact cells were subjected to protease treatment (Fig. 1 B). Digestion of surface-localized receptor is evident both in
terms of loss of receptor protein and by the production of
a protease digestion product corresponding to the protected, intracellular CTD plus final transmembrane domain (Davis et al., 1993
; the antibody used for the immunoprecipitation is STE3 CTD specific). Wild-type Ste3p in
such a regimen is only transiently susceptible to the added proteases, reflective of its short surface residency time
(Davis et al., 1993
). Here, we find that the
413-468 mutant receptor remains susceptible to the proteases even 90 min after synthesis (Fig. 1 B). Quantitation via PhosphorImaging indicates that 75% of
413-468 receptor protein
is susceptible to protease 45 min after synthesis and 60%
remains susceptible at the 90-min time point. Cell integrity
is maintained throughout the digestion protocol as evidenced by the complete protection from digestion of a cytoplasmic marker protein, phospho-glycerol kinase (Pgk1p;
Fig. 1 B, bottom panel). We conclude from its prolonged
surface residency time that turnover defect found for
Ste3
413-468p does indeed reflect impaired endocytosis.
Deletional Analysis of the Sequences Required for Rapid Endocytosis and Turnover
To identify the sequence element(s) that direct the constitutive endocytosis of Ste3p we have constructed a finer series of deletions within the 413-468 interval. First we
tested the effects of three deletions extending progressively in from the receptor COOH terminus. The in vivo
rate of turnover was assessed for wild-type Ste3p and for
each mutant receptor via a pulse-chase regimen similar to
that described for Fig. 1 A. Immunoprecipitated receptors
were subjected to SDS-PAGE (Fig. 2 A) and then to PhosphorImaging analysis to quantitate their rate of loss to
degradation (Fig. 2 B). As seen previously (Fig. 1 A), turnover is rapid for wild-type Ste3p and wholly blocked for
Ste3413-468p. The two intermediate deletion mutants,
450-468 and
441-450 show intermediate rates of turnover. Quantitation of the receptor-associated radioactivity
at each time-point is shown in Fig. 2 B. Most of the data
points for each receptor conform to a linear plot, allowing
the calculation of turnover half-life for each mutant (Fig. 2
B). For wild-type Ste3p, a t1/2 of 16 min is calculated. For
Ste3
450-468p, turnover remains rapid, though somewhat
less so than wild-type: a t1/2 of 21 min is calculated. With
the progressive extension of these deletions, a progressive
impairment is evident. A t1/2 of 65 min is calculated for the
441-468 receptor and the turnover block appears to be complete for the
413-468 receptor.
For two of the receptors, namely wild-type and 450-
468, the 90-min time-points fail to conform to the line defined by the other time-points (Fig. 2 B). This is seen consistently and indicates that a minority of these receptors,
3% for wild-type and 13% for Ste3
450-468p, do not participate in the normal endocytic turnover mechanism. Hypothetically, these could be receptors in the process of undergoing recycling or receptors that are trapped to a locale that prohibits their endocytosis.
Fig. 3 summarizes the results from a total of eight
COOH-terminal deletion mutants. Again, it is apparent
that endocytic function is lost gradually with progressive
removal of sequences from the COOH-terminal end. A
small, but significant slowing of the turnover rate is apparent with removal of the COOH-terminal 12 amino acids (Fig. 3; t1/2 of 25 min for 458-468 vs. 15 min for wild-type). A second transition is broached with the
447-468
deletion; the t1/2 for turnover slows from 25 to 40 min. Further impairment is seen when the deletion is extended to
residue 441, with the t1/2 for
441-468 being 70 min. Finally, with the deletion extended to residue 423, endocytosis appears to be wholly blocked. This gradual loss of function for deletions extending over the COOH-terminal 48 amino acids indicates the "signal" for constitutive endocytosis may be large and complex, potentially composed of
redundant functional elements.
To delimit the upstream boundary of this endocytosis
signal, small internal deletions were introduced into the
450-468 mutant receptor. The
450-468 deletion removes the COOH-terminal 20 amino acid residues and
was used as the context for these other small deletions to
eliminate the potential contribution of redundant elements within the 450-470 interval. The experiment of Fig.
1 indicated that a large portion of the receptor CTD (the
320-414 interval) is not required for rapid endocytic turnover. Again here within the
450-468 context, introduction of a second deletion of residues 398-414 caused no
additional defect; the t1/2 for turnover of the double deletion mutant
398-414, 450-468 was unchanged from that
of the parental
450-468 construct (Fig. 3). However, introduction of either the
413-424 or the
423-434 deletions into the
450-468 receptor wholly blocked turnover.
This indicates that sequences required for rapid endocytosis extend to the NH2-terminal side of residue 423, potentially as far as residue 413. Thus, the 36-residue-long 414-
449 sequence interval appears to be the most critical for
directing rapid endocytosis. Consistent with this, deletion
of just these amino acids (STE3
413-451) abolishes turnover (Fig. 3). However, the COOH-terminal 20 residues
also may contribute to turnover: both the
450-468 and
458-468 deletions have small, but significant effects on
the turnover rate (Figs. 2 and 3). Support for the participation of the COOH-terminal 20 residues in endocytosis is
offered below (see Fig. 9) with experiments indicating a
functional redundancy between sequence elements in the
414-449 interval and those in the 450-470 interval.
Sequences That Direct Receptor Endocytosis Also Direct Receptor Ubiquitination
We next tested if the STE3 mutants defective for constitutive endocytosis are also defective for the associated constitutive ubiquitination. Receptor ubiquitination levels of
the various STE3 mutants were assessed via Western analysis: ubiquitinated receptor species manifest an 8-kD shift
with each added ubiquitin moiety. Detection of the ubiquitinated forms is improved with the use of several key experimental conditions, including receptor overproduction
(~10-fold) from the GAL1 promoter. This enhances detection of the ubiquitinated forms, yet neither alters receptor turnover rate nor the proportion of the receptor subject to ubiquitination (data not shown). Secondly, to avoid
loss of the ubiquitinated forms to vacuolar degradation,
pep4 mutants deficient in vacuolar protease activity were
used. Finally, treatment of protein extracts with phosphatase before electrophoresis eliminates the substantial heterogeneity in gel mobility because of the heterogenous
phosphorylation of Ste3p (Fig. 7; also Roth and Davis,
1996
).
|
|
In Fig. 4 A, the ubiquitination of three receptor mutants
is compared with wild-type. For wild-type, the majority
species (~80%) has no attached ubiquitin (Roth and
Davis, 1996). The remaining 20% distributes almost
equally between two, more slowly-migrating, ubiquitinated forms (indicated in Fig. 4 A, arrows at left), the mono- and di-ubiquitinated receptor species (Roth and
Davis, 1996
). Mutants that block endocytosis, namely
413-468 and
413-451 (Fig. 3), also blocked receptor
ubiquitination (Fig. 4 A). Furthermore, the
450-468 mutant receptor that showed diminished turnover (Fig. 2)
also shows diminished ubiquitination (Fig. 4 A). Quantitation of these results along with similar analyses applied to
the other mutant receptors is reported in Fig. 3. Comparison of the turnover half-life with ubiquitination levels
shows a consistent correlation between the two
further
compelling evidence for a functional connection linking
ubiquitination and endocytosis.
Previously, we have shown that in end4-1 cells, constitutive Ste3p endocytosis is blocked and the receptor is consequently trapped at the cell surface in a ubiquitinated
state (Roth and Davis, 1996). Here, we again make use of
the end4 mutant strain background to eliminate endocytosis as a possible contributing cause to the differences seen
in ubiquitination levels for the different receptor mutants.
If, for instance, ubiquitin addition occurred at some post-surface, endosomal step, then mutations like
413-468,
which block uptake, might block ubiquitination by preventing access of the receptor to downstream steps. In
such an experiment (Fig. 4 B), essentially the same result
is seen as was apparent in the END4+ background (Fig. 4
A). While both the wild-type and
450-468 receptors are
ubiquitinated, the
413-468 receptor is not, and the
441-
468 receptor shows greatly reduced levels of ubiquitination. Ste3p ubiquitination, therefore, likely occurs while
receptor is surface localized, serving potentially as the initiating trigger for uptake.
Sufficiency of the Signal for Ubiquitination and Endocytosis
We have identified a domain mapping to the COOH-terminal portion of the receptor's CTD that directs both the
ubiquitination and the rapid constitutive endocytosis of
the receptor. In this section we test the sufficiency of this
sequence to direct the ubiquitination and endocytosis of a
normally long-lived constituent of the plasma membrane,
the plasma membrane ATPase Pma1p. Like Ste3p, Pma1p
is a polytopic plasma membrane protein with a cytoplasmically exposed, COOH-terminal tail. Unlike Ste3p,
Pma1p is quite stable with a t1/2 estimated at >11 h (Benito
et al., 1991). By site-directed mutagenesis, an XhoI site
was created at the penultimate PMA1 codon to provide a
locus for the in-frame fusion of the various STE3 sequences to be tested. Fusions are expressed from the
GAL1 promoter, and have embedded near the NH2-terminal-encoding end of the PMA1 ORF, a single HA epitope
tag that does not impair PMA1 function (Harris et al.,
1994
). Thus, turnover of the fusion proteins may be easily
assessed as follows: (a) PMA1-STE3 proteins are expressed via growth on galactose-containing medium, (b)
synthesis of the construct protein is then blocked with the
addition of glucose, and (c) the time-dependent loss is followed via immunoblotting with anti-HA mAb. This same
experimental system was used previously to show that a
108-residue portion of Ste6p sufficed to direct endocytosis
of a PMA1-STE6 fusion protein (Kolling and Losko, 1997
).
Five different PMA1-STE3 fusion proteins were constructed, each with a different segment of the STE3 CTD fused at the COOH terminus of PMA1. The parental HA-tagged Pma1p showed no evidence of turnover during the 2-h course of this experiment (Fig. 5). Attachment of the 347-398 STE3 sequence interval was without effect; this PMA1-STE3 fusion protein shows a stability equivalent to that of the starting PMA1 construct (Fig. 5). In contrast, attachment of the 400-470 interval, encoding the COOH-terminal 71 amino acids of Ste3p, does destabilize the resulting PMA1-STE3 fusion protein (Fig. 5). This fusion protein is degraded with a t1/2 of 40 min (Table II). Likewise, the fusion carrying the 400-449 STE3 sequence interval also turns over (Fig. 5); a t1/2 of 50 min is calculated for this protein (Table II). Turnover slows dramatically when the critical 414-449 interval is violated as it is for PMA1-STE3(400-440) (Fig. 5); a t1/2 of 90 min is calculated (Table II). Finally, removal of the entire 414-449 interval, as for PMA1-STE3(400-412), effectively abolishes turnover (Fig. 5; Table II).
|
In addition to the five PMA1-STE3 constructs described above, four additional constructs were made with the STE3 contribution extending various distances into the critical 414-449 interval. These constructs were subjected to analyses identical to those in Fig. 5 and the resulting calculated turnover t1/2 is reported for each (Table II). In general, these results correlate quite well with the role of these sequences in Ste3p turnover (Fig. 3). Sequences that showed an intermediate rate of turnover in the Ste3p context also show intermediate effects when tested in the PMA1-STE3 context. In addition, we find that the minimal endocytic signal, the 414-449 STE3 interval, suffices to promote turnover of the resulting PMA1-STE3 fusion (Table II).
Degradation of Ste3p depends on transport from the
cell surface to the vacuole (Davis et al., 1993; Roth and
Davis, 1996
). Is the turnover of the PMA1-STE3 constructs instigated by a similar route of transport? To test
involvement of vacuolar proteases in PMA1-STE3 fusion
turnover, constructs with the STE3 400-449 peptide sequence interval were introduced into isogenic PEP4+ and
pep4
strains and turnover assessed. While turnover proceeds in the PEP4+ background, in the pep4
context,
turnover is completely blocked (Fig. 6, left panel). This is
also true for the longer PMA1-STE3(400-470) fusion protein (data not shown). Thus, like wild-type Ste3p, turnover of these PMA1-STE3 fusions also depends on vacuolar
proteases and therefore also likely depends on transport
to the vacuole.
|
To test the dependence of turnover on endocytic transport, an end4 mutant was used. The PMA1-STE3(400- 449) fusion was introduced into isogenic END4+ and end4-1 cells. Turnover is blocked in the end4 background (Fig. 6, right panel), indicating that as for Ste3p itself, turnover of the PMA1-STE3(400-449) fusion depends on its endocytosis from cell surface to vacuole. Again, this is also true for the PMA1-STE3(400-470) fusion as well (data not shown).
Also apparent in the end4 background is the presence of
modified forms of the 400-449 PMA1-STE3 fusion that
migrate more slowly than the mature fusion protein band
(Fig. 6, species indicated by arrowhead). Although accentuated by end4, a similar modification of the PMA1-STE3(400-449) fusions was apparent both in the wild-type strain backgrounds (Fig. 5) and in the pep4 background
(Fig. 6, left panel). Such modified species also were seen
for the longer PMA1-STE3(400-470) fusion, but were not
seen either for the other fusions or for the HA-tagged
Pma1p alone (Fig. 7 B).
To test if the modification is ubiquitination, we have
made use of a myc epitope-tagged ubiquitin (Ellison and
Hochstrasser, 1991). Multi-copy yeast plasmids expressing
either wild-type ubiquitin, the myc-tagged ubiquitin, or no
ubiquitin were introduced into end4 cells with one of three
PMA1-STE3 constructs, the 400-470 fusion, the 400-449
fusion, or the long-lived 400-412 fusion. The PMA1-STE3
fusion proteins were purified from extracts of these cells
via immunoprecipitation with the HA mAb. Immunoblotting of the purified fusion proteins with the HA mAb, allowed visualization of the fusion constructs and showed
that overexpression of either the wild-type or epitope-tagged ubiquitin results in no obvious change in the
PMA1-STE3 fusion protein electrophoretic mobility (Fig.
7 A). Parallel immunoblots, probed instead with the myc mAb, allowed assessment of the incorporation of the myc-tagged ubiquitin into the fusion constructs (Fig. 7 A). The
stable 400-412 fusion, did not react with the anti-myc monoclonal (Fig. 7 A). In contrast, both the 400-449 and 400-
470 fusions showed a clear reaction. Reaction was specific
for cells coexpressing the myc epitope-tagged ubiquitin.
The 400-470 fusion showed a somewhat stronger reaction
than did the 400-449 fusion (Fig. 7 A), consistent with a
greater proportion of this fusion protein being ubiquitin modified.
Using our panel of PMA1-STE3 fusions, we next tested the STE3 sequence requirement for the ubiquitin modification. Extracts prepared from end4 cells expressing each of the different PMA1-STE3 fusions were subjected to Western analysis with the HA mAb (Fig. 7 B). A prominent accumulation of modified forms is apparent for the two fusions retaining the critical 414-449 interval, i.e., the 400-449 and 400-470 fusions (Fig. 7 B). Smaller fusions violating the integrity of 414-449 STE3 interval do not show this massive accumulation of modified species (Fig. 7 B). (The minor species migrating slightly above the major fusion protein band for each of these other fusions is not likely to represent ubiquitinated protein: the 400-412 fusion that manifests this minor species in Fig. 7 B, fails to react with myc mAb in Fig. 7 A.) Thus, just as truncation of the 414-449 interval impairs Ste3p ubiquitination, intrusion into this interval also abolishes ubiquitination of the corresponding PMA1-STE3 fusion proteins. We conclude that the 414-449 STE3 sequence interval is the minimal signal that suffices to specify both ubiquitination and endocytosis.
Mutational Analysis of the Endocytosis Signal
The 36-residue-long 414-449 peptide sequence comprises
a ubiquitination/endocytosis signal for Ste3p. To gain insight into the nature of this sequence, we have applied alanine-scanning mutagenesis across this entire interval.
Again, to avoid the potential effects of redundant signal
elements within the 450-470 interval, mutations were
made within the STE3450-468 context. 12 mutants in total spanning the 414-450 interval were constructed (Fig. 8). For each, three adjacent residues were substituted by
three alanines. Each mutant was subjected to pulse-chase
analysis identical to that previously applied to the various
STE3 deletion mutants (Figs. 1 and 2). The calculated t1/2
are reported in Fig. 8.
Results from the alanine scanning show a good concordance with the deletion analysis (Fig. 3). While the deletion analysis indicated a requirement of the entire 414-449
interval, the 414-433 interval was found to be particularly
crucial. For instance, though grossly impaired, the 441-
468 and
434-468 receptor mutants still showed a slow
rate of turnover (t1/2 = 70 min; Fig. 3). Deletions that violated the more critical 414-433 interval (e.g.,
423-468 or
413-468) completely abolished turnover. Likewise for
the triple alanine mutations, mutations that totally disabled endocytosis were concentrated within the crucial
414-433 interval; of the five mutants with t1/2 of >2 h, four
locate within the 414-433 interval (Fig. 8). Alanine mutations in the 434-450 interval generally showed intermediate levels of impairment (Fig. 8). The one exception to this
is the replacement of the Ser-Ser-Asn 438-440 tripeptide,
which appeared to fully disable endocytosis. We are presently without an explanation of how this triple alanine replacement could have a more severe phenotype than the
deletion interval encompassing it.
The number of these triple alanine mutations having pronounced effects on turnover is striking (Fig. 8). Residues important for endocytosis distribute throughout the 414-450 interval. No singular sequence motif is discernible. Instead, the entire 36-residue-long sequence is required in its totality. The most prominent feature of this interval, which we later discuss, is the high proportion of acidic (glutamates and aspartates) and hydroxylated residues (serines and threonines).
Redundant Elements within the 450-470 STE3 Interval
The 414-449 sequence is the minimal ubiquitination/endocytosis signal. This, however, does not exclude the possible participation of other STE3 sequences. A small, but
reproducible effect on turnover was seen, for instance,
with removal of the COOH-terminal 20 amino acids: the
t1/2 for Ste3450-468p is 25 min compared with 15 min for
wild-type (Fig. 2). Furthermore, like the 414-449 interval, the 450-470 interval is also rich in both acidic and hydroxylated amino acids. To test the contribution of these sequences to endocytosis, we have examined several mutations that were known to disable turnover in the
450-468
receptor, within the context of the full-length receptor.
The triple alanine replacements were constructed and
tested originally within the context of the 450-468 deleted receptor. Two of these that had disabling effects in
the
450-468 receptor, were chosen and reconstructed in
the full-length receptor context. The effects on the rate of
receptor turnover were tested via pulse-chase analysis in
both contexts (Fig. 9). In the
450-468 context, both mutations severely retard endocytic turnover. Where the
450-468 receptor turns over with a t1/2 of 25 min, the
same receptor with the triple alanine substitution of the
Thr-Leu-Asp tripeptide at 426-428 turns over with a t1/2 of
90 min (Fig. 9). The effect of the alanine substitution of
the 417-419 tripeptide Ile-Ser-Leu is even more severe,
showing a t1/2 of >2 h (Fig. 9). In the full-length receptor
context, while the two triple alanine mutations do cause some slowing of turnover, the effects are far less severe.
While the wild-type receptor turns over with a t1/2 of 12 min in this experiment, for triple alanine substitutions at
417-419 and at 426-428, turnover is slowed only to a t1/2 of
21 and 17 min, respectively (Fig. 9). We conclude therefore that elements within the 450-470 sequence may compensate the loss of key elements in the 414-449 interval.
We have seen previously that extension of a COOH-terminal deletion from residue 450 to residue 441 severely
impairs endocytic turnover (Figs. 2, 3, and 9; compare
450-468 with
441-468). This implies a critical role for
the 441-449 sequence. To test the importance of these sequences in an otherwise full-length receptor, we have constructed a receptor mutant in which only the 441-451 sequences are deleted. Like the two triple alanine mutants
tested above, the
441-451 deletion has only minor effects in the full-length receptor context (Fig. 9). Again this suggests a functional redundancy between elements in the
414-449 interval with elements in the COOH-terminal
450-470 interval. It may be that the entire 57-residue-long
segment from 414 to the COOH terminus normally functions to specify Ste3p ubiquitination and endocytosis. The
414-449 interval, highlighted by our deletion analysis may correspond simply to that minimal portion still capable of
promoting rapid endocytosis.
![]() |
Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-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. 3, compare 450-468 and
447-468). With further extension to residue 441 or to residue 434, the t1/2 increased to 70 min (Fig. 3,
441-468 and
434-468). Only
deletions extending to residue 423 and residue 413 effectively abolished endocytosis (Fig. 3,
423-468 and
413-
468). Thus it appears that both the rate of endocytosis, and
also the degree of ubiquitin modification (Fig. 3) 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. 8) suggested the possibility that
Lys432 might serve as the sole ubiquitin acceptor site
within the 450-468 receptor. However, this is not supported by the effects of a K432R point mutation. Within
the context of the
450-468 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.
![]() |
Footnotes |
---|
Received for publication 27 February 1998 and in revised form 5 June 1998.
Address all correspondence to N.G. Davis, Departments of Surgery and Pharmacology, Wayne State University School of Medicine, Elliman Building, room 1205, 421 E. Canfield, Detroit, MI 48201. Tel.: (313) 577-7807. Fax: (313) 577-7642. E-mail: ndavis{at}cmb.biosci.wayne.eduWe thank T. Stevens for the PGK-specific antiserum and J. Haber for the HA-tagged GAL1-PMA1 plasmid.
This work was supported by a research grant from the National Science Foundation (MCB 95-06839). D.M. Sullivan was supported by a postdoctoral research fellowship from the National Institutes of Health (T32- GM08420).
![]() |
Abbreviations used in this paper |
---|
CTD, cytoplasmically disposed, COOH-terminal domain; ORF, open reading frame.
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