From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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G protein-coupled receptors that transduce
signals for many hormones, neurotransmitters, and inflammatory
mediators are internalized and subsequently recycled to the plasma
membrane, or down-regulated by targeting to lysosomes for degradation.
Here we have characterized yeast G protein-coupled receptors
(GPCRs)1 function in signal
transduction pathways that allow eukaryotic cells to respond to sensory stimuli and extracellular signals, including various hormones and
neurotransmitters. In response to agonist stimulation, GPCRs are
regulated by phosphorylation, sequestration, and down-regulation, which
limit the strength or duration of physiological responses (1, 2).
Whereas down-regulation persistently attenuates signaling by targeting
GPCRs for lysosomal degradation, sequestration apparently serves to
reactivate desensitized receptors by allowing them to be
dephosphorylated and recycled in active form to the cell surface (3,
4).
Recent studies have begun to elucidate the mechanisms governing the
sequestration and down-regulation of GPCRs (2). For example,
GPCRs activated by peptide mating pheromones in the yeast
Saccharomyces cerevisiae have provided insights into the
molecular mechanisms of receptor down-regulation (7). Agonist binding triggers phosphorylation and subsequent ubiquitination of yeast Yeast pheromone receptors transit the endocytic pathway through the
action of several gene products. For example, a small GTP-binding
protein (Ypt5/Vps21) and an NSF homolog (Sec18), regulate fusion or
maturation of early endosomes (11, 17), and two syntaxin homologs, Tlg1
and Tlg2, are required for receptor down-regulation (18).
Here we have used yeast cells expressing green fluorescent protein
fused to the Materials, Media, and Isotopes--
Enzymes used for recombinant
DNA methods were purchased from commercial sources and used according
to the suppliers' recommendations. Sources of growth media for yeast
and bacterial cells have been described (19).
[35S]Na2SO4 (carrier-free) was
obtained from ICN. 7-Aminochloromethylcoumarin (CMAC) and monoclonal
antibodies specific for carboxypeptidase Y were obtained from Molecular Probes.
Yeast Strains and Plasmids--
The S. cerevisiae
strains used in these studies were JE115 (MATa
ura3-52 leu2-3, 112 his3 Mutagenesis and Genetic Screening--
Transposon insertion
mutagenesis and a visual screen were performed to identify cells that
displayed aberrant localization of Ste2-GFP. Two pools of a
transposon-mutagenized yeast genomic DNA library (21) cleaved with
NotI were transformed into JE115 cells carrying the
STE2-GFP fusion on a centromeric plasmid. Approximately 6,000 transformants were treated individually with pheromone (1 µM
To identify genes affected in the mutants isolated from the visual
screen, chromosomal DNA was prepared, digested with EcoRI, HindIII, or BamHI, which cleaves once in the
transposable element and cleaves in the adjacent chromosomal DNA (21).
Fragments containing the bla gene from the transposable
element and flanking yeast genomic DNA were cloned into pCRScriptCAM
SK(+) (Stratagene) cleaved with EcoRI, HindIII,
or BamHI. These plasmids were introduced into
Escherichia coli by selecting for resistance to ampicillin and chloramphenicol. Plasmid DNA was prepared and sequenced using a
primer that anneals to the Tn3 38 base pair repeat at the left end of
the transposon; this identified the chromosomal locus disrupted by the
transposable element.
Pheromone Response Assays--
Quantitative bioassays (halo
assays) were used to measure the apparent sensitivity of cells to
pheromone-induced growth arrest (22). The relative responsiveness of
cells was determined by comparing the amount of Ligand Binding and Receptor Internalization Assays--
Methods
used to purify [35S]
Endocytosis of the Immunoblotting--
Cultures were grown to a density of 2 × 107 cells/ml in synthetic medium (SD-uracil) to select
for plasmids pVAM3.416 and pvam3-6.416. Immunoblotting methods used to detect carboxypeptidase Y (CPY) in yeast
whole-cell extracts were similar to those previously described (24).
Cells were lysed by mechanical disruption in Laemmli sample buffer, and
the protein concentration of the cleared lysates was determined by the
Bradford method and adjusted to 1 mg/ml with Laemmli sample buffer
prior to SDS-polyacrylamide gel electrophoresis.
Fluorescence Microscopy--
Yeast cell cultures were labeled
with CMAC at a concentration of 100 µM for 15 min in
synthetic medium at room temperature to examine vacuole morphology. To
visualize
To detect changes in the distribution of receptors upon treating cells
with pheromone, we mixed MATa cells (JE115) expressing
GFP-tagged receptors (from pRS314STE2-GFP) with a 4-fold excess of MAT Characterization of GFP-tagged Wild Type and Truncated
Tagging full-length Ste2-GFP Localizes to the Plasma Membrane and Putative Endocytic
Compartments--
We subsequently examined the localization of
full-length Ste2-GFP in the absence or presence of mating pheromone.
Consistent with our previous immunofluorescence studies using fixed
cells expressing myc-tagged receptors (19), Ste2-GFP was
present at the cell surface (Fig.
2A), particularly in daughter
cells, probably due to polarization of the secretory pathway. Ste2-GFP
was also detected at the tips of cell surface projections induced
2 h after exposing cell cultures to pheromone (data not shown),
similar to what has been reported using untagged receptors
(27).
Ste2-GFP also localized to two morphologically distinct types of
intracellular vesicular structures. Smaller vesicles (usually >5/cell;
Fig. 2A, single arrowhead) were
present throughout the cytoplasm. The second type of vesicular
structures were larger (Fig.
2A, double
arrow), less numerous (typically 2-5/cell), and nearly
always located next to the vacuole, which was stained with the vital
dye CMAC (28).
Two approaches were used to determine whether the intracellular
vesicles observed in cells expressing Ste2-GFP were secretory or
endocytic. First, we examined the localization of Ste2
Second, because receptor internalization is stimulated by pheromone, we
determined whether the number of intracellular vesicles increases when
cells are exposed to the pheromone secreted by a neighboring cell of
opposite mating type. For these experiments, MATa
cells expressing Ste2-GFP and MAT Ste2-GFP Localizes to the Vacuole Lumen--
Although Ste2-GFP was
expected to be associated with the plasma membrane, cell surface
projections, and endocytic compartments, it was surprising that
Ste2-GFP-derived fluorescence was also present in the lumen of the
vacuole (Fig. 2A, top row). GFP
accumulated in the vacuole of most cells, although the level differed
significantly from cell to cell (also see Fig. 3, top
row), possibly reflecting different extents of basal
internalization of Ste2-GFP, rates of GFP degradation, or plasmid copy
number. Localization to the vacuole lumen was confirmed by co-staining
with CMAC.
Accumulation of Ste2-GFP or its degradation products in the vacuole
lumen was unexpected because the GFP domain was fused to the
cytoplasmic tail of the receptor, which should remain cytoplasmically disposed if endocytic trafficking to the vacuole exclusively involves fusion events between unilamellar donor and acceptor membranes. The
potential significance of this observation with respect to the
mechanisms of endocytic transport is presented under
"Discussion."
Identification of Vam3, a Vacuolar t-SNARE That Affects the
Localization of Ste2-GFP--
To identify gene products that
participate in endocytic trafficking of
Mutants were identified in the following way. Pools of a
transposon-mutagenized yeast DNA library were transformed into a ste2 null mutant (JE115) expressing Ste2-GFP from a
centromeric plasmid; this eliminates the possibility that localization
of Ste2-GFP could be affected by the presence of untagged receptors expressed from the chromosome. Transformants were screened individually in the absence and presence of pheromone for alterations in receptor distribution. Two transformants displayed a similar aberrant pattern of
Ste2-GFP localization, either in the absence or presence of pheromone
(data not shown). In contrast to wild type cells, these mutants
displayed intense fluorescence in numerous punctate intracellular patches or vesicles (Fig. 3, second and fourth
rows). Furthermore, both mutants possessed fragmented
vacuoles (as revealed by CMAC staining). In contrast, when these
mutants expressed endocytosis-defective receptors, Ste2
Analysis of transposon insertion sites revealed that both mutants
carried disruptions of the VAM3/PTH1 gene, which encodes a
protein of 283 amino acids with similarity to yeast and mammalian syntaxins or t-SNAREs (20, 24, 29, 30). In one mutant (Fig. 3,
second row) the insertion occurred at codon 112, near sequences encoding the second predicted coiled-coil domain of Vam3; this was termed the vam3-100 allele. In the second
mutant (Fig. 3, fourth row) the insertion
occurred at codon 268, within sequences encoding the transmembrane
domain of Vam3; this was termed the vam3-101 allele. These
mutations appeared to cause the mutant phenotypes because introduction
of the wild type VAM3 gene on a centromeric plasmid
corrected the defects in Ste2-GFP localization and vacuolar morphology
(Fig. 3, third and fifth rows). Interestingly,
however, the wild type VAM3 plasmid only partially corrected
the phenotype of the vam3-101 mutant, because some cells in
the population exhibited a wild type distribution of Ste2-GFP and
intact vacuoles whereas others had a mutant phenotype (Fig. 3,
fifth row). Thus, a mutation truncating Vam3 near
its transmembrane domain may be partially dominant-negative. Similar truncations of other SNARE proteins are dominant-negative (31, 32).
vam3 Mutations Preserve the Signaling Activity, Expression, Ligand
Binding Affinity, and Internalization of Evidence That Vam3 Is Required to Deliver Receptors to the
Vacuole--
Because VAM3 is dispensable for receptor
expression, signaling, and internalization, it may be required
specifically for the targeting and/or fusion of late endocytic vesicles
with the vacuole. Consistent with this hypothesis, previous studies
have shown that Vam3 localizes to the vacuole membrane where it is
required for homotypic vacuole-vacuole fusion, autophagy, and targeting
of biosynthetic transport vesicles that deliver carboxypeptidase Y and
alkaline phosphatase to the vacuole (20, 24, 29, 30). However, the
apparent mislocalization of Ste2-GFP in vam3 mutants could
be due to fragmentation of the vacuole, a phenotype of vam3 mutants. Therefore, it was important to determine whether Vam3 is
directly involved in the targeting or fusion of receptor-bearing endocytic vesicles to the vacuole.
Accordingly, we examined the kinetics with which untagged receptors are
targeted to the vacuole by following the rate that receptor-internalized 35S-labeled
A block in
As an alternative means of determining whether defects in the
degradation of receptor-internalized
The rates of 35S-labeled Ste2-GFP as a Marker of Endocytic Trafficking in Living
Cells--
To study G protein-coupled receptor trafficking and
down-regulation in yeast, we have utilized cells that express green
fluorescent protein fused to the
Surprisingly, cells expressing Ste2-GFP accumulate GFP in the vacuole
lumen, even though GFP has been fused to the cytoplasmic C-terminal
tail of the receptor. Lumenal accumulation of GFP requires receptor
internalization because GFP fluorescence is not detected in the vacuole
lumen when cells express an endocytosis-defective form of Ste2-GFP, or
GFP alone (38).
Although the mechanisms responsible for lumenal accumulation of GFP
remain to be determined, we suggest two possibilities that would allow
both the extracellular and cytoplasmic domains of the receptor to gain
access to the lumen of the vacuole (Fig. 7). In one model, endocytic transport
vesicles bearing Ste2-GFP are engulfed by an acceptor compartment, such
as the vacuole. Alternatively, the membranes of late
endosomes/prevacuoles or vacuoles bearing Ste2-GFP could involute and
pinch off into the lumen. This is consistent with previous studies
showing that Ste2 and internalized gold particles are present in
multivesicular bodies (11, 12). An engulfment or involution mechanism
may be an important step in the receptor down-regulation pathway
because either would provide a means of degrading bound Role of Vam3, a Vacuolar t-SNARE Homolog, in Receptor
Down-regulation--
By using a visual screen to identify mutants
defective in the localization of Ste2-GFP, we obtained recessive and
partially dominant mutations in the VAM3 gene.
VAM3 was first identified in a screen for mutants displaying
fragmented vacuoles (39) and subsequently has been shown to encode a
vacuole membrane-localized syntaxin (t-SNARE) homolog required for
three processes: docking and fusion of autophagic and biosynthetic
transport vesicles to the vacuole (20, 24, 30, 40), and homotypic
fusion of vacuolar membranes in vitro (29).
Our studies suggest that Vam3 is required for endocytic trafficking of
Although vam3 null mutants are defective in a late step in
the receptor down-regulation pathway, they appear to be normal with
respect to pheromone response, receptor polarization to the tips of
cell surface projections and mating. This is expected because receptor
internalization is unaffected in vam3 mutants. Thus, even in
vam3 mutants receptors are effectively internalized and
sequestered from the cell surface.
Whereas yeast mating pheromone receptors are down-regulated upon
agonist exposure, mammalian GPCRs can be internalized and subsequently
recycled to the plasma membrane, or down-regulated and degraded. For
example, -factor receptors tagged with green
fluorescent protein (Ste2-GFP) and used them to obtain mutants
defective in receptor down-regulation. In wild type cells, Ste2-GFP was
functional and localized to the plasma membrane and endocytic
compartments. Although GFP was fused to the cytoplasmic tail of the
receptor, GFP also accumulated in the lumen of the vacuole, suggesting
that the receptor's extracellular and cytoplasmic domains are degraded
within the vacuole lumen. Transposon mutagenesis and a visual screen
were used to identify mutants displaying aberrant localization of
Ste2-GFP. Mutants that accumulated Ste2-GFP in numerous intracellular
vesicles carried disruptions of the VAM3/PTH1 gene, which
encodes a syntaxin homolog (t-SNARE) required for homotypic vacuole
membrane fusion, autophagy and fusion of biosynthetic transport
vesicles with the vacuole. We provide evidence that Vam3 is required
for the delivery of
-factor receptor-ligand complexes to the
vacuole. Vam3 homologs in mammalian cells may mediate late steps in the
down-regulation and lysosomal degradation pathways of various G
protein-coupled receptors.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-arrestin acts as an adaptor that binds GPCRs and clathrin (5),
recruiting receptors into coated pits that pinch off by a
dynamin-dependent mechanism (6). In contrast, the molecular mechanisms governing the transport of GPCRs along subsequent steps of
the endocytic pathways responsible for sequestration or down-regulation are less well understood.
-factor receptors on their cytoplasmic C-terminal domains (8, 9).
Ubiquitinated receptors are internalized by processes that involve
clathrin, actin, myosin, and fimbrin (10), and subsequently transported
through vesicular intermediates similar to early and late endosomes in
mammalian cells (11, 12). In late endosomal/prevacuolar compartments,
pheromone receptor trafficking is thought to converge with biosynthetic
pathways that deliver precursors of resident vacuolar proteins
(13-15). Subsequent events transport pheromone-receptor complexes to
the vacuole where they are degraded by vacuolar hydrolases (11,
16).
-factor receptor (Ste2-GFP) as a new tool to localize
receptors in compartments of the endocytic pathway and to identify gene
products involved in receptor down-regulation. We describe evidence
that a syntaxin homolog encoded by the VAM3 gene is required
for delivery of Ste2 to the vacuole, a late step in the down-regulation pathway.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-1 trp1
ste2
::LEU2 sst1-
5; Ref. 8),
KBY49 (vam3-100 derivative of JE115), KBY62
(vam3-101 derivative of JE115), KBY58 (ste2
derivative of JE115), and KBY65 (vam3
derivative of
KBY58). The unmarked ste2
allele of KBY58 was introduced
by two-step gene deletion using JE115 and ClaI-cut YIp5ste2
(19), and confirmed by loss of LEU2.
The VAM3 coding region was deleted using
ApaI/SacI-cut
pvam3::LEU2 (20), to create KBY65. To
localize
-factor receptors in living cells, we constructed plasmid
pRS314STE2-GFP, which expresses green fluorescent protein
(GFP) fused to wild type receptors at their extreme C termini.
Polymerase chain reaction was used to generate GFP(S65T) coding
sequences flanked by BamHI restriction sites. This
polymerase chain reaction product was digested with BamHI
and cloned into pGEX2T (Amersham Pharmacia Biotech) that had been cut
with BamHI to create pGEX2T-GFP. Plasmid
pRS314STE2-GFP was created by inserting a BamHI
fragment containing the GFP (S65T) coding region in the appropriate
reading frame from pGEX2T-GFP into the BglII site of
pRS314STE2
ter in which the wild type termination codon
was inactivated (19). To fuse GFP to
-factor receptors lacking their
C-terminal cytoplasmic tails, we inserted the BamHI fragment containing the GFP(S65T) coding region in the appropriate reading frame
from pGEX2T-GFP into the BglII site of
pRS314ste2-300ter (19), to create plasmid
pRS314ste2
tail-GFP. To express VAM3, plasmid
pRS313VAM3 was constructed by isolating a 1.8-kilobase pair
XmnI-ApaI fragment encompassing the
VAM3 locus from pVAM3.416 (20), and inserting it
into pRS313 that had been cleaved with SmaI and
ApaI. The vam3-6 temperature-sensitive allele
was expressed from pvam3-6.416 (20).
-factor, 3 h) and screened using fluorescence
microscopy for abnormal distribution of Ste2-GFP.
-factor required to
form a halo 20 mm in diameter, as indicated by interpolating
dose-response curves. Quantitative mating assays were performed as
described (22).
-factor and perform ligand binding
assays with inviable, intact cells have been described (19). Assays of
cells expressing wild type or GFP-tagged receptors employed [35S]
-factor (30 Ci/mmol) at concentrations ranging
from 1 to 10 nM. Ligand binding data were plotted according
to the method of Scatchard and fitted by nonlinear least mean square
regression. Nonspecific binding was determined in the presence of a
500-fold excess of unlabeled
-factor.
-factor receptor was determined by measuring the
rates that cells internalize bound 35S-labeled
-factor,
as described previously (23). Rates of degradation of internalized
35S-labeled
-factor by wild type cells or
vam3 mutants were determined as described previously (11,
23), with the following modifications. Cells were grown at 26 °C or
30 °C to a density of 107 cells/ml, washed, and
suspended in YPB (10 mM PIPES, pH 6.0, 1 mM
MgCl2, 0.1 mM EDTA, 1% yeast extract, 2%
bactopeptone). 35S-Labeled
-factor (1 µCi, 100 nM final concentration) was bound to cells for 1 h
under conditions that inhibit ligand-induced internalization of
receptors (YPB at 0 °C). Unbound ligand was removed by pelleting
cells, and cells were suspended in YPD medium, pH 6.0, at 26 °C, or
38 °C if vam3 temperature-sensitive mutants were
analyzed. Receptor internalization was allowed to proceed. Aliquots
(100 µl) were removed at various times and diluted into 10 ml 50 mM sodium citrate buffer, pH 1.1, containing 10 mM KF on ice to remove surface-bound
-factor. Cells were
filtered, washed with 50 mM potassium phosphate buffer, pH
6.0, containing 10 mM NaN3 and 10 mM KF, and suspended in extraction buffer (23). Internalized 35S-labeled
-factor extracted from cells
was subjected to thin layer chromatography to resolve intact and
degraded
-factor species, as described previously (23).
-factor receptors, cultures were grown to a density of
2 × 107 cells/ml in synthetic medium (SD-tryptophan)
to select for plasmids that express GFP-tagged receptors. Cells were
harvested by centrifugation, suspended in low fluorescence medium (25),
and observed under an Olympus epifluorescence microscope equipped with
UG-1, BP490, and BP545 dichroic filters and a cooled CCD camera (Dage).
cells (RK537-3B) to provide a source of
-factor. The cell suspension (2 µl) was placed on an agarose pad
containing medium as described previously (25). Cells were imaged at
room temperature on an Olympus epifluorescence microscope equipped with
stage and shutter controllers. Time lapse fluorescence images were
collected by acquisition of 10 focal planes (0.5 µm apart), and
collapsed into a single two-dimensional image as described previously
(25).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-Factor
Receptors--
To characterize the endocytic trafficking of yeast
-factor receptors and to identify mutants defective in this process,
we needed a rapid and reliable means of examining receptor
localization. Accordingly, we constructed a centromeric plasmid that
expresses green fluorescent protein (GFP, S65T) fused to the extreme
C-terminal cytoplasmic tail of the
-factor receptor, Ste2-GFP (Fig.
1). We also constructed a similar plasmid
that expresses GFP fused to a truncated
-factor receptor lacking its
cytoplasmic C-terminal tail, Ste2
tail-GFP, which should not be
internalized from the cell surface (8, 26). These two forms of Ste2-GFP
were expressed from the STE2 promoter in a ste2
null mutant and characterized with regard to signaling activity,
agonist binding affinity, cell surface expression, and internalization
kinetics.
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Fig. 1.
Receptor binding, expression, signaling, and
internalization properties. A, agonist binding
affinities and cell surface receptor expression levels of cells (JE115)
expressing wild type receptors from pRS314STE2
(Ste2) or full-length GFP-tagged receptors from
pRS314STE2-GFP (Ste2-GFP), and of an isogenic
vam3-100 mutant (KBY49) expressing full-length GFP-tagged
receptors from pRS314STE2-GFP (vam3-100). The
results were obtained using two independent transformants assayed in
duplicate; standard errors for Kd and
Bmax values were less than 15%. B,
pheromone response of cells (JE115) expressing wild type receptors from
pRS314STE2 (open circles) or full-length
GFP-tagged receptors from pRS314STE2-GFP (closed
circles), a vam3-100 mutant (KBY49) expressing
full-length GFP-tagged receptors from pRS314STE2-GFP
(open squares), and a vam3-101 mutant (KBY62)
expressing full-length GFP-tagged receptors from
pRS314STE2-GFP (closed squares). Assays of
pheromone-induced growth arrest were used. The data presented are the
average of two independent experiments; standard deviations were less
than 10%. C, internalization of
[35S] -factor by cells (JE115) expressing wild type
receptors from pRS314STE2 (open circles),
full-length GFP-tagged receptors from pRS314STE2-GFP
(closed circles) or tail-truncated GFP-tagged receptors from
pRS314ste2
tail-GFP (closed squares), and a
vam3-100 mutant (KBY49) expressing full-length GFP-tagged
receptors from pRS314STE2-GFP (open squares).
Receptor internalization is indicated by the percentage of prebound
[35S]
-factor internalized over time. Each data point
represents the average of two independent assays performed in
duplicate, except for cells expressing Ste2p
tail-GFP, which were
analyzed once; standard errors are indicated.
-factor receptors with GFP did not appear to
affect receptor signaling, agonist binding affinity, cell surface
expression, or internalization. Tagged and untagged receptors displayed
similar agonist binding affinities (Kd = 4.9 and 5.3 nM for Ste2-GFP and Ste2, respectively; Fig. 1A)
and cell surface expression levels (Bmax = 15,000 and 19,000 sites/cell respectively, Fig. 1A). Cells
expressing Ste2-GFP responded to pheromone with nearly normal
efficiency (Fig. 1B) and mated as efficiently as cells
expressing untagged receptors (data not shown). Furthermore, cells
expressing Ste2-GFP internalized receptors at a rate indistinguishable
from that of cells expressing full-length untagged receptors, as
indicated by measuring rates of 35S-labeled
-factor
uptake (Fig. 1C). Cells expressing receptors lacking their
cytoplasmic C-terminal domains (Ste2
tail-GFP) were defective in this
assay (Fig. 1C) and conferred a pheromone supersensitive phenotype identical to untagged truncated receptors (data not shown),
as expected (8, 26).
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Fig. 2.
Localization of GFP-tagged -factor
receptors. A, localization of Ste2-GFP in the absence
of pheromone. Cells (JE115) expressed Ste2-GFP from
pRS314STE2-GFP (top row) or Ste2
tail-GFP from
pRS314ste2
tail-GFP (bottom row) to visualize
receptors (GFP), and were stained with CMAC to visualize
vacuoles (CMAC). Putative early endosomes (single
arrowhead) and late endosomal/prevacuolar compartments
(double arrowhead) are indicated. B, changes in
the localization of Ste2-GFP upon pheromone stimulation. Time-lapse
fluorescence video microscopy of individual cells incubated at 22 °C
was used to monitor changes in the distribution of Ste2-GFP in
MATa cells (JE115; indicated by asterisks
in the Nomarski image in the lower right panel) in response
to the pheromone secreted by neighboring MAT
cells
(RK537-3B; unlabeled in the Nomarski image and invisible in the
fluorescence images because they did not express a GFP-tagged protein).
At each time point, 10 z-axis fluorescence images (0.5 µm
apart) were acquired and collapsed into a single image, allowing the
number of endosomes containing Ste2-GFP in each cell to be counted.
Over the indicated time interval, the number of early and/or late
endosomes containing Ste2-GFP increased 2-4-fold.
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Fig. 3.
Localization of GFP-tagged -factor
receptors in wild type cells and vam3 mutants. Wild
type and mutant cells expressed Ste2-GFP from pRS314STE2-GFP
to visualize receptors (GFP) and were labeled with CMAC to
visualize vacuolar morphology (CMAC); Nomarski images of
stained cells are shown (DIC). The strains used were JE115
(wild type), KBY49 (vam3-100), and KBY62
(vam3-101). Plasmid pRS313VAM3 (CEN
VAM3) expressing the wild type VAM3 gene was introduced
into the indicated mutants.
tail-GFP, which fails to undergo endocytosis. Ste2
tail-GFP was present at the
cell surface, but it did not localize to intracellular vesicular
structures (Fig. 2A). A small proportion of Ste2
tail-GFP also localized to a perinuclear ring similar to the endoplasmic reticulum, suggesting that this fusion protein has a slight
biosynthetic defect. This ring structure was not the vacuole membrane
because it did not stain with CMAC.
cells that do not express a GFP-tagged protein were mixed and mounted on a
medium-containing agarose pad. Time-lapse fluorescence video microscopy
was used to follow the distribution of Ste2-GFP in single cells over
time. Consistent with an endocytic origin, the number of vesicles
containing Ste2-GFP increased 2-4-fold as cells responded over time to
the pheromone produced by neighboring cells of opposite mating type (Fig. 2B). Increases in vesicle number were detected 20 min
after cells were first exposed to pheromone, before receptor expression is induced. Results of these two experiments, and previous
immunofluorescence studies of pheromone receptors (11, 14, 15),
indicate that small vesicles containing Ste2-GFP are probably early
endosomes, and the larger vesicles are late endosome/prevacuolar compartments.
-factor receptors, we used a
visual screen to isolate mutants in which the intracellular
distribution of Ste2-GFP was abnormal. Transposon insertion mutagenesis
was used because insertion sites are easily determined with the aid of
the completed yeast genome sequence. However, this method is likely to
reveal only a subset of the genes involved in endocytic trafficking
because transposon insertions occur at nonrandom sites, insertions into essential genes cannot be recovered, and subtle but significant differences in receptor localization could be overlooked in a visual screen.
tail-GFP,
they lacked fluorescence in intracellular vesicles (data not
shown), suggesting that the localization of full-length Ste2-GFP within
these vesicles requires receptor endocytosis. Thus, the mutants
displayed defects in vacuolar morphology and/or targeting of endocytic
vesicles containing Ste2-GFP to vacuoles.
-Factor
Receptors--
Several observations suggested that the
vam3-100 and vam3-101 mutations preserve
receptor expression, signaling and internalization. First, wild type
cells and vam3-100 mutants expressed receptors having
similar agonist binding affinities (4.9 and 6.2 nM,
respectively) and cell surface expression levels
(Bmax = 14,000 and 15,000 sites/cell, respectively) (Fig. 1A). Second, vam3-100 and
vam3-101 mutants expressing Ste2-GFP responded to pheromone
with efficiencies similar to wild type cells expressing Ste2-GFP (Fig.
1B) and mated with normal efficiency (data not shown).
Third, rates of receptor internalization were unaffected in
vam3-100 mutants expressing Ste2-GFP, as revealed by
measuring rates of 35S-labeled
-factor uptake (Fig.
1C).
-factor is degraded,
which requires active vacuolar hydrolases (9). Initially, these
experiments were performed using wild type cells and
vam3-100 and vam3-101 mutants. As shown in Fig.
4, wild type cells expressing untagged
full-length receptors rapidly internalized and degraded
35S-labeled
-factor. After internalization was
initiated,
-factor degradation products appeared within 30 min, and
the levels of intact
-factor decreased with an apparent
t1/2 of ~30 min, consistent with previous
reports (9). In vam3-100 and vam3-101 cells
(Fig. 4), both processes (loss of intact internalized
-factor and
accumulation of
-factor degradation products) were essentially
blocked. As expected, these defects were corrected in
vam3-100 mutants bearing the wild type VAM3 gene
on a centromeric plasmid (Fig. 4).
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Fig. 4.
Effects of vam3 null mutations on
degradation of receptor-internalized -factor. Degradation of
receptor-internalized [35S]
-factor was followed over
time as described under "Experimental Procedures." The initial time
point (t = 0) was taken 10 min after internalization
was initiated, before degradation occurred. Wild type receptors were
expressed from pRS314STE2 in wild type cells (JE115),
vam3-100 cells (KBY49), vam3-101 cells (KBY62),
and vam3-100 cells (KBY49) carrying the wild type
VAM3 gene on a plasmid (pRS313VAM3).
A, resolution of internalized [35S]
-factor
by thin layer chromatography and detection by autoradiography; intact
and partially degraded
-factor are indicated. Results of a
representative experiment are shown. B, quantitation of
-factor degradation rates. Wild type receptors were expressed from
pRS314STE2 in wild type cells (JE115; open
circles), vam3-100 cells (KBY49; closed
circles), vam3-101 cells (KBY62; open
squares), and vam3-100 cells (KBY49) carrying the wild
type VAM3 gene on a plasmid (pRS313VAM3;
closed squares). Data obtained from two independent
experiments were analyzed by densitometry, averaged, and expressed as a
percentage of intact
-factor present in cells at the initial time
point. Standard errors are indicated.
-factor degradation seen in vam3 mutants could
be caused by a defect in targeting of endocytic vesicles to the vacuole, or by a defect in the proteolytic processing and activation of
vacuolar hydrolases required to degrade
-factor. To address the
latter possibility, we used immunoblotting to examine the steady state
levels of a mature vacuolar protease, mCPY, in wild type and
vam3 mutant cells. The results indicated that the
vam3-100 and vam3-101 mutations had relatively
little effect on steady state levels of mCPY (Fig.
5).
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Fig. 5.
Expression levels of mature carboxypeptidase
Y in wild type cells and vam3 mutants. Steady state
levels of mature carboxypeptidase Y (mCPY) were determined by
immunoblotting using equivalent amounts of extracts from JE115
(wild type, lane 1), KBY49
(vam3-100, lane 2), KBY62
(vam3-101, lane 3), KBY58 (wild
type, lane 4), KBY65 (vam3 ,
lane 5), KBY65 carrying the vam3-6
allele on plasmid pvam3-6.416 (vam3-6,
lane 6), and KBY65 carrying the wild type
VAM3 gene on plasmid pVAM3.416 (VAM3,
lane 7). Monoclonal antibodies specific for CPY
and an enhanced chemiluminescence detection system were used.
-factor are due directly to a
targeting defect, we analyzed a vam3 temperature sensitive mutant (vam3-6) (20). This allele of VAM3
produces a protein product that is rapidly inactivated (within 10 min)
upon temperature shift. Therefore, growth of cells at the permissive
temperature (26 °C) should allow vacuolar hydrolases to accumulate
to normal levels, and, after a brief shift to the nonpermissive
temperature (38 °C),
-factor uptake and degradation assays can be
performed. However, this requires that the levels of mature CPY are
unaffected after a brief incubation (15 min) of vam3-6
mutants at the nonpermissive temperature (38 °C). This prerequisite
was fulfilled, as revealed by immunoblotting experiments (Fig. 5).
-factor degradation in wild
type and vam3-6 mutant cells were measured following a
15-min shift to the nonpermissive temperature. As shown in Fig.
6, wild type cells expressing untagged
full-length receptors internalized and degraded 35S-labeled
-factor at 38 °C. Degradation products began to accumulate 30 min
after internalization was initiated, and increased over time as intact
-factor disappeared (Fig. 6). The kinetics of
-factor degradation
in wild type cells shifted to 38 °C were somewhat slower
(t1/2 of ~50 min) than in previous
experiments, probably because the actin cytoskeleton, which is required
for receptor internalization, is depolarized transiently at elevated temperatures (33). Nevertheless, at the nonpermissive temperature,
-factor degradation was ~3-fold slower in the vam3-6
mutant (t1/2 >90 min) than in wild type cells,
and a more severe defect was observed in vam3
mutants
(Fig. 6). The residual degradation of
-factor that occurs in the
vam3-6 mutant is probably carried out by partially
processed or misprocessed peptidases that accumulate in prevacuolar
compartments at the nonpermissive temperature (20). As expected, these
defects in
-factor degradation were corrected by introducing the
wild type VAM3 gene on a centromeric plasmid (Fig. 6).
Receptor degradation is probably also blocked in vam3 mutants because previous investigations have indicated that
-factor and its receptor transit the same pathway to the vacuole (7, 34).
Therefore, the results of our study suggest that Vam3 has a direct role
in the down-regulation pathway by targeting or fusing receptor-bearing
endocytic vesicles with the vacuole.
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Fig. 6.
Effects of a vam3
temperature-sensitive mutation on degradation of
receptor-internalized -factor. A, resolution of
internalized [35S]
-factor by thin layer chromatography
and detection by autoradiography; intact and partially degraded
-factor are indicated. Wild type receptors were expressed from
pRS314STE2 in wild type cells (JE115; wild type),
and in a vam3
mutant (KBY65) carrying an empty vector
(pRS416; vam3
+ vector), a plasmid expressing the
vam3-6 temperature-sensitive allele
(pvam3-6.416; vam3
+ CENvam3-6), or a
plasmid expressing the wild type VAM3 gene
(pVAM3.416; vam3
+ CENVAM3). Results of a
representative experiment are shown. B, quantitation of
-factor degradation rates. Wild type receptors were expressed from
pRS314STE2 in wild type cells (JE115; open
circles), and in a vam3
mutant (KBY65) carrying an
empty vector (pRS416; closed circles), a plasmid expressing
the vam3-6 temperature-sensitive allele
(pvam3-6.416; open squares) or a plasmid
expressing the wild type VAM3 gene (pVAM3.416;
closed squares). Data obtained from two independent
experiments were analyzed by densitometry, averaged, and expressed as a
percentage of intact
-factor present in cells at the initial time
point. Standard errors are indicated.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-factor receptor, Ste2-GFP. Similar
to studies of mammalian GPCRs tagged with GFP (35, 36), we find that Ste2-GFP exhibits essentially normal agonist binding affinity, cell
surface expression, and internalization rates. Moreover, the
distribution of Ste2-GFP at the plasma membrane in naive or pheromone-treated cells is similar to that of untagged or
myc-tagged receptors (19, 27). Ste2-GFP also localizes to
endosomes and prevacuolar compartments, which upon pheromone
stimulation increases in accord with faster rates of receptor
internalization (37).
-factor and
the extracellular and intracellular domains of the receptor.
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Fig. 7.
Hypothetical mechanisms whereby GFP derived
from Ste2-GFP accumulates in the vacuole lumen. GFP-tagged
-factor receptors (Ste2-GFP), and various compartments in the
endocytic pathway are indicated. See "Discussion" for
details.
-factor receptors to the vacuole. We find that in vam3
null mutants or vam3-6 temperature-sensitive mutants
shifted briefly to the nonpermissive temperature, the proteolytic
degradation of
-factor internalized by receptors is strongly
inhibited. Because vam3 mutants do not have significant
deficits in the levels of mature hydrolases within the vacuole, we
suggest that Vam3 has a direct role in the targeting of vesicles
bearing receptor-ligand complexes to the vacuole. Furthermore, because
Vam3 is required for biosynthetic transport of vacuolar hydrolase
precursors, our findings reinforce the current view that endocytic
trafficking of receptor-ligand complexes converges at a prevacuolar
step with pathways that deliver immature soluble hydrolases to the vacuole.
2-adrenergic receptors can be internalized and
recycled in response to short term agonist stimulation, whereas thrombin receptors are down-regulated (41). Understanding how these
different fates are achieved requires determining which endocytic
compartments are used by both the recycling and down-regulation pathways, and which compartments are used uniquely by each pathway. As
suggested by our analysis of vam3 mutants and studies of
other SNARE mutants (31, 32), it may be possible to define the
compartments transited by recycled versus down-regulated
GPCRs in mammalian cells by using dominant-negative forms of various
syntaxins to block endocytic transport at specific steps. We anticipate
that homologs of Vam3 in mammalian cells will be required for the
down-regulation and degradation of various GPCRs.
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ACKNOWLEDGEMENTS |
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We thank T. Darsow and S. Emr for providing strains and plasmids, M. Goebl for the transposon insertion library, M. Overton for constructing KBY58, and M. Linder and members of our laboratory for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM44592 (to K. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Division of Cellular & Molecular Medicine, Howard
Hughes Medical Institute, School of Medicine, University of
California, San Diego, La Jolla, CA 92093-0668.
§ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-1668; Fax: 314-362-7463; E-mail: kblumer{at}cellbio.wustl.edu.
The abbreviations used are: GPCR, G protein-coupled receptor; CMAC, 7-aminochloromethylcoumarin; CPY, carboxypeptidase Y; G protein, guanine nucleotide-binding regulatory protein; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; SNARE, soluble NSF attachment protein receptor.
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REFERENCES |
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