(Received for publication, December 21, 1995; and in revised form, January 31, 1996)
From the
Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. This permease undergoes endocytosis and subsequent degradation in cells subjected to adverse conditions. The data presented here show that uracil permease also undergoes basal turnover under normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubiquitin variants have been used to show that uracil permease is ubiquitinated in vivo. The ubiquitin-permease conjugates that are readily demonstrated in wild type cells were barely detectable in npi1 mutant cells, indicating that uracil permease may be a physiological substrate of the Npi1p ubiquitin ligase. The lack of ubiquitination of the permease in npi1 cells resulted in an increase in active, i.e. plasma membrane-located, permease, suggesting that there is a direct relationship between ubiquitination and removal of the permease from the plasma membrane. The accumulation of ubiquitin-permease conjugates in thermosensitive act1 mutant cells, deficient in the internalization step of endocytosis is consistent with this idea. On the other hand, the degradation of uracil permease does not require a functional proteasome since the permease was not stabilized in either pre1 pre2 or cim3 and cim5 mutant cells that have impaired catalytic (pre) or regulatory (cim) proteasome subunits. In contrast, both basal and stress-stimulated turnover rates were greatly reduced in pep4 mutant cells having defective vacuolar protease activities. We therefore propose that ubiquitination of uracil permease acts as a signal for endocytosis of the protein that is subsequently degraded in the vacuole.
Yeast cells have developed many regulated transport systems that
respond sensitively to changes in the extracellular environment. Saccharomyces cerevisiae has several amino acid and sugar
permeases that undergo down-regulation at both the transcriptional and
post-translational levels when a preferred nutrient becomes available (1, 2) . Providing cells grown on a poor nitrogen
source with NH ions blocks expression of
the general amino acid Gap1 permease and any existing Gap1 is rapidly
inactivated(3) . This latter phenomenon, called nitrogen
catabolite inactivation, has recently been shown to be accompanied by
the proteolytic breakdown of the Gap1 permease(4) . The maltose
transporter undergoes similar dual regulation, including proteolysis
triggered by glucose under nitrogen starvation conditions(5) .
The degradation of ITR1 encoded transporter is triggered by
adding its own substrate, inositol(6) . Control of permease
stability therefore appears to be a general process, which provides a
rapid way of turning off the uptake of unneeded nutrients. The
molecular mechanism responsible for switching between the stability and
degradation of these plasma membrane proteins remains poorly
understood.
We have used uracil permease to examine the degradative pathway of yeast permeases. This permease, encoded by the FUR4 gene, is delivered to the plasma membrane via the secretory pathway (7) and is phosphorylated on serine residues in a post-Golgi compartment, probably at the plasma membrane(8) . Permease is rapidly degraded under several adverse conditions, such as the approach of the stationary growth phase or the inhibition of protein synthesis. Its degradation follows its endocytosis, as it is dependent on the products of the END3 and END4 genes(9) , required for endocytosis of pheromone receptors(10) . The permease accumulates in the vacuole of growing pep4 mutant cells, deficient in the activities of the main vacuolar proteases(9) . This suggests that there is a constitutive turnover in addition to that triggered by stressful conditions.
Uracil permease contains a 9-residue sequence similar to
the ``destruction box'' of mitotic cyclins. This box appears
to be essential for the conjugation of ubiquitin (Ub) ()to
cyclins and subsequent proteolysis (11, 12) . A
permease having a point mutation (R294A) within this region is
resistant to stress-induced degradation(13) . We have therefore
suggested that ubiquitination is a signal for permease degradation, as
it is for the breakdown of the mitotic cyclins. Ub-conjugates formation
requires the combined action of three classes of enzymes: the
Ub-activating enzyme (E1), Ub-conjugating enzymes (UBC, or E2), and at
least in some cases Ub-protein ligases (E3), which play a role in
substrate recognition(14, 15) . The hypothesis that Ub
is conjugated to the uracil permease is supported by the finding that
the permease degradation triggered by stressful conditions requires the NPI1 gene product(4) , which is structurally and
functionally related to E6-AP, a human Ub-protein
ligase(4, 16) .
The data presented here demonstrate that uracil permease indeed undergoes Npi1p-dependent ubiquitination. We show that permease ubiquitination is a cell surface event, followed by endocytosis and vacuolar proteolytic breakdown. We therefore propose that ubiquitination of uracil permease signals its endocytosis.
Figure 1:
Stabilization of uracil permease in npi1 mutant cells. 23344C (WT) and 27038a (npi1) strains transformed with p195gF were grown to an A of 0.7 with galactose as a carbon source.
Cells were then labeled by incubation for 10 min with
[
S]methionine in the growth medium and chased
with methionine for the indicated times (in min). A, uracil
permease was immunoprecipitated and analyzed by SDS-PAGE and
fluorography. B, PhosphorImager analysis quantitation of this
gel and other gels from independent experiments.
, wild type;
, npi1 cells.
Figure 2:
Steady-state levels of uracil permease in
growing wild type and npi1 cells. A, 23344C (WT) and 27038a (npi1) cells harboring YEp352fF were
grown to an A of 0.7 (lanes 1 and 2) or 1.3 (lanes 3 and 4). Protein extracts
were prepared, resolved by electrophoresis, and blotted onto a
nitrocellulose filter. The blot was probed with both uracil permease
and plasma membrane (H
)-ATPase antibodies. The
molecular masses of the markers are given in kDa. Permease appears as a
broad band corresponding to different phosphorylated bands(8) .
The resolution of these different species vary depending on the gels. B, uracil uptake activity (nmol/min/A
)
of the same cells measured during the growth phase.
, wild type;
, npi1.
Figure 3:
Ubiquitination of uracil permease. A, 27061b cells bearing plasmids p195gF (lane 1),
YEp105 (lane 6), or YEp112 (lane 7) or both p195gF
and either YEp112 (lane 2), YEp96 (lane 3), or YEp105 (lanes 4 and 5) were grown in the presence of
CuSO (except for lane 5), to induce normal or
tagged Ub synthesis from the CUP1 promoter. + above the lanes indicates the overexpression of the corresponding
proteins; u corresponds to a low production of Myc-tagged Ub
from the uninduced CUP1 promoter. Crude membranes were
prepared from cells collected in mid-log phase and analyzed by Western
blot with uracil permease-specific antibodies. The molecular masses of
the markers are given in kDa. B, membrane extracts were
precipitated with anti-uracil permease antibodies and further analyzed
by Western blot with mouse anti-HA (lanes 1-3) or
anti-Myc (lanes 4-7) monoclonal antibodies. The
indications above the lanes are as in A.
The Ub-permease conjugates in npi1 mutant cells and wild type cells were compared by Western blot analysis of plasma membrane-enriched fractions (Fig. 4). The high molecular mass species visible in parental cells (lane 1), identified as Ub-conjugates, were essentially absent from membrane-extracts from npi1 cells (lane 2). Even membranes derived from npi1 cells overexpressing HA-tagged Ub, which led to an enrichment of ubiquitinated species in parental cells (lane 3), showed no high molecular mass species (lane 4). Glucose-grown npi1 cells, which overexpressed uracil permease from its endogenous promoter, also lacked Ub-permease conjugates (not shown). It is therefore very probable that Npi1p is directly involved in the normal ubiquitination of the permease and thus contributes to its instability.
Figure 4: Lack of Ub-permease conjugates in npi1 cells. 23344C (WT) and 27038a (npi1) cells transformed with p195gF (lanes 1 and 2); 27061b and 27064b (their trp1 counterparts) cotransformed with p195gF and YEp112 (lanes 3 and 4) were collected in mid-log phase and used to prepare membranes. Uracil permease was visualized by Western blotting. The loads were adjusted to obtain roughly equal intensities of the main permease signals from wild type and npi1 cells.
A functional cytoskeleton
is essential for the internalization step of receptor-mediated
endocytosis(31, 32) . A strain carrying a conditional
thermosensitive mutation in actin, act1-1, was shown to
have an especially rapid and complete defect in the internalization of
-factor linked to its receptor, as soon as the temperature was
raised to 37 °C(32) . The act1 and parental cells
were transformed with multicopy plasmid encoding galactose-inducible
permease. Permease activity, corresponding to plasma membrane-arrived
protein, was induced at the same rate in wild type and mutant cells
grown at the permissive temperature (data not shown). The turnover of
uracil permease was analyzed in act1 and parental cells at
both permissive and restrictive temperatures after inhibition of
protein synthesis by cycloheximide. Uracil uptake was measured at
various time intervals, and protein extracts were prepared and analyzed
for uracil permease by immunoblots. As expected, inhibition of protein
synthesis triggered the loss of uracil uptake and parallel permease
degradation in wild type cells, and both events were accelerated when
the temperature was raised from 24 °C to 37 °C (Fig. 5).
At the restrictive temperature, and to a lesser extent at the
permissive temperature, the act1 mutation provided strong
protection against stress-induced loss of uracil uptake, and permease
degradation. Permease, which was rapidly degraded in wild type cells at
37 °C, was maintained in act1 cells over 2 h (Fig. 5B). Thus for uracil permease, as for
-factor, internalization, hence subsequent degradation, were
greatly impaired in act1 mutant cells at the restrictive
temperature and were partially defective at the permissive temperature.
Figure 5:
Defective internalization of uracil
permease in act1 cells submitted to inhibition of protein
synthesis. NY13 (WT) and NY279 (act1) strains
transformed with the plasmid p195gF were grown at 24 °C to an A of 0.6 with galactose as carbon source. Cells
were transferred or not, to 37 °C, and cycloheximide (100
µg/ml) was added to the medium 10 min later. A and B, protein extracts were prepared at the times indicated,
after the addition of cycloheximide, and analyzed for uracil permease
by Western immunoblotting (A, 24 °C; B, 37
°C). C, uracil uptake was measured at different times
after addition of cycloheximide. Results are percent of initial
activities.
, wild type at 24 °C;
, wild type at 37
°C;
, act1 at 24 °C;
, act1 at
37 °C.
Some Ub-permease conjugates were observed in total extracts from act1 cells grown at 24 °C (Fig. 5A, lane 5). The extent of ubiquitination was further analyzed
using plasma membrane-enriched fractions from act1 and
parental cells grown at 24 °C and incubated (or not) for 10 min at
37 °C with cycloheximide. Fig. 6shows an immunoblot probed
with anti-(H)-ATPase and uracil permease antibodies.
Whereas wild type and act1 mutant cells exhibited the same
amount and pattern of the abundant, stable (H
)-ATPase,
they differed considerably in their pattern of permease species.
Growing act1 cells contained more Ub-permease conjugates
relative to the main permease signal than wild type cells (Fig. 6, lanes 1 and 2). Incubation with
cycloheximide for 10 min at 37 °C led to a further enrichment in
Ub-permease conjugates in act1 cells (lane 4),
whereas there was a noticeable decrease in all permease specific
signals in wild type cells (lane 3). Thus, both basal and
stress-stimulated permease degradation involved the formation of
Ub-permease conjugates at a step preceding permease internalization.
Figure 6:
Ub-permease conjugates accumulate in act1 cells. NY13 (WT) and NY279 (act1) cells
transformed and grown as described above were collected before (lanes 1 and 2) or after (lanes 3 and 4) incubation for 10 min at 37 °C with cycloheximide. They
were used to prepare plasma membrane-enriched fractions. Aliquots
containing the same amount of proteins were separated by SDS-PAGE and
analyzed by immunoblotting for uracil permease and
(H)-ATPase. Brackets indicate Ub-permease
conjugates.
The cim3, cim5, and pre1 pre2 mutant and isogenic wild type strains were
cotransformed with multicopy plasmids encoding galactose-inducible
uracil permease and Ub-proline--galactosidase fusion protein
(Ub-Pro-
-gal), an artificial substrate of the Ub proteolytic
pathway(25) . Exponentially growing cells were labeled for 10
min with Tran
S-label. Chase was then initiated by adding
methionine plus cysteine, a condition that leads to a stimulated
turnover of uracil permease (see ``Materials and Methods'').
The half-life of uracil permease was essentially the same in each of
these proteasome mutants and in the corresponding wild type cells (Fig. 7). In contrast, the short-lived Ub-Pro-
-gal, which
was rapidly degraded in wild type cells, remained almost constant
during the whole chase, indicating that the proteasome function was
indeed blocked in these mutant cells under our experimental conditions.
A pulse-chase experiment was performed under the same conditions using pep4 and parental cells expressing galactose-inducible
permease (Fig. 8). Permease was far more stable (about 5-fold)
in pep4 cells than in wild type cells. These data indicate
that stress-induced permease turnover depends on vacuolar proteolysis
and that it is independent of the proteasome.
Figure 7:
Stress-stimulated permease turnover is
independent of the proteasome. A and B, WCG4a (WT) and WCG4-11/22a (pre1 pre2) cells
cotransformed with pgF and p(Ub-Pro--gal) were grown at 30 °C
with galactose as a carbon source to mid log phase, and then incubated
for 1 h at 37 °C. Cells were labeled at 37 °C with
Tran
S-label for 5 min and chased with methionine plus
cysteine for the times indicated. Samples were immunoprecipitated with
anti-uracil permease and anti-
-gal-specific antibodies.
Immunoprecipitated proteins were resolved by SDS-PAGE, and the data
were quantified by PhosphorImager analysis.
, wild type;
, pre1 pre2. C and D, a pulse-chase analysis
was performed as described in A, except that temperature was 30 °C,
on YPH499 (WT,
), CMY763 (cim 3,
), and
CMY765 (cim5,
) cells.
Figure 8:
Stress-stimulated permease turnover is
dependent of vacuolar proteases. A pulse-chase experiment was performed
as described in the legend of Fig. 7on W303-1B/D (WT, ) and IW-6A (pep4,
) cells
transformed with p195gF.
Similarly, mutations
in the proteasome and the vacuolar proteolytic activities had different
effects on basal permease degradation. The steady-state levels of
uracil permease in the proteasome mutants were the same as in wild type
cells, while these mutant cells accumulated Ub-Pro--gal (data not
shown). In contrast, uracil permease accumulated in exponentially
growing pep4 cells(9) , while there was almost no
accumulation of the (H
)-ATPase (Fig. 9). We
have already shown by immunofluorescence that the accumulated permease
is located within structures overlapping with the vacuoles, visualized
by Nomarski optics(38) . Western blot analysis of the
essentially vacuolar permease from total extracts of pep4 cells gave identical patterns of permease species when
immunodetection was performed with specific N-terminal or C-terminal
antipeptide antibodies (Fig. 9). Therefore, uracil permease did
not appear to undergo partial proteolytic processing on its way from
the plasma membrane to the vacuole, an observation which confirmed that
the proteasome is not involved in permease degradation. Most of the
vacuole-accumulated protein appeared to be non-ubiquitinated. Although
an artifactual de-ubiquitination during preparation of protein extracts
cannot be entirely excluded, this observation indicated more likely
that permease underwent de-ubiquitination within endocytic
compartments, or after its delivery to the vacuole.
Figure 9:
Uracil permease accumulates in pep4 cells as an entire protein. W303-1B/D (WT) and
IW-6A (pep4) cells transformed with pfF were grown with
glucose as a carbon source. Protein extracts were prepared from
exponentially growing cells withdrawn at an A of
0.6. Proteins from 0.2 ml of culture were analyzed by Western blots
using N-terminal permease antipeptide antibody. The nitrocellulose was
then treated as described by the manufacturer (Amersham), in order to
obtain the complete retrieve of the immunodetected signal, and probed
simultaneously with C-terminal antipeptide antibody, and
[H
]-ATPase
antibody.
The above data indicate that uracil permease constitutively undergoes a moderate turnover, besides stress-stimulated turnover. Both basal and stress-stimulated permease turnover involve a Npi1p-dependent ubiquitination step, which is required prior to permease endocytosis and vacuolar degradation.
Ubiquitination of uracil permease was found strikingly reduced in a npi1 mutant, deficient in the Npi1p/Rsp5p Ub-protein ligase. Despite extensive knowledge of the ubiquitin system(14) , few genes are known to encode Ub-protein ligases: the human gene encoding E6-AP (39) and the yeast UBR1 and NPI1/RSP5 genes(16, 40) . The Npi1p protein, conserved from yeast to man, has a putative phospholipid interaction motif (C2) in its N terminus, followed by several repeats of a WWP domain (41) (also referred as WW; (42) ), which has recently been implicated in mediating protein-protein interactions(43) . The C-terminal domain of Npi1p is homologous to the hect domain of the human E6-AP(4, 16, 41) . If the activity of Npi1p/Rsp5p as a Ub ligase has been documented in vitro(16) , our results provide strong evidence that Npi1p might act in vivo as the Ub ligase involved in the ubiquitination of uracil permease. Npi1p and uracil permease would then be one of the few Ub-protein ligase/substrate pairs identified so far and, to our knowledge, the first example of a pair Ub ligase/plasma membrane protein. Given the protection against ammonium-induced proteolytic breakdown of Gap1p observed in npi1 mutant cells(4) , it is highly probable that Npi1p/Rsp5p also functions as the Ub ligase required for a putative ubiquitination of Gap1p.
Little is known about the signals required for ubiquitination of cytosolic and, moreover, membrane-bound proteins. The N-end rule-based degradation signal is associated with the involvement of Ubr1 ligase, and a physiological target of this ligase has been identified in yeast(44) . The ``destruction box'' of mitotic cyclins is also a signal for ubiquitination and subsequent degradation(11) . Uracil permease has a sequence similar to the cyclin destruction box, and replacement of its invariant arginine by an alanine (R294A permease) shows that degradation of uracil permease proceeds by destruction box-dependent pathway under stress conditions (13) . In contrast, the basal turnover in growing cells might be destruction box-independent, as it is not affected in the R294A permease (data not shown). As the stability of the uracil permease depends upon the metabolic state of the cells, its ubiquitination might be regulated. There is a change in the phosphorylation of the permease in parallel with a change in turnover ( (45) and data not shown), suggesting that, as reported for a few proteins(46, 47) , the extent of phosphorylation might influence the conjugation of Ub to the permease and its subsequent instability. On the other hand, modulations of the Ub system might influence the extent of permease ubiquitination. The yeast Ub system is activated in cells faced with various adverse conditions(15) , which also lead to an increase in the permease turnover.
Two
independent lines of evidence indicate that the ubiquitination of
uracil permease is a cell-surface event. First, permease is stabilized
at the cell-surface in npi1 mutant cells, that are deficient
in permease ubiquitination. Secondly, ubiquitinated forms of uracil
permease accumulate in act1 mutant cells, that are deficient
in the internalization step of endocytosis. Both observations apply to
basal, as well as stress-stimulated permease degradation. These data
demonstrate that ubiquitination is a necessary event prior to permease
internalization. Similar conclusions have been reached for three other
yeast plasma membrane proteins, two ABC transporters Ste6p and Pdr5p,
and Ste2p, the -factor receptor, which also accumulate in
ubiquitinated forms in an early endocytosis
mutant(48, 49, 50) . Activation-induced
multi-ubiquitination of the mammalian immunoglobulin E receptor is also
a cell surface event(51) .
Multi-ubiquitinated soluble proteins are thought to be recognized and degraded by the proteasome(33, 52) . It was demonstrated recently that the proteasome is also involved in the degradation of ubiquitinated membrane-bound proteins located in the endoplasmic reticulum(53, 54) . The present results show that the half-life of the plasma membrane uracil permease is unchanged in several catalytic and regulatory mutants of the proteasome, while its degradation is strongly impaired in a pep4 mutant, deficient in vacuolar protease activities. Uracil permease does not undergo any partial proteolytic cleavage before its delivery to the vacuole since it accumulates in the vacuole of pep4 cells as an entire protein. The turnover of uracil permease therefore appears to be exclusively vacuolar, a conclusion that extends out the knowledge relative to the proteolytic breakdown of the few yeast plasma membrane proteins known to be ubiquitinated(48, 49, 55) .
The present data indicate that the ubiquitination of uracil permease functions as a signal targeting permease for internalization and subsequent degradation. Ligand-induced ubiquitination also signals Ste2p for endocytosis(49) . While ubiquitination and subsequent vacuolar degradation have been formally demonstrated for only a very few yeast plasma membrane proteins to date(48, 49, 50) , the degradation of several other transporters is known to depend on E2 or E3 enzymes(4, 56) . It is thus likely that conjugation to Ub, and subsequent endocytosis, is a general process enabling the regulated degradation of a number of plasma membrane proteins in yeast in response to various environmental cues. Whether specific machinery is involved in the recognition and internalization of ubiquitinated membrane proteins is one of the questions open for future investigation. Studying the intracellular fate of uracil permease should provide new insights into the signals, steps, and molecules involved in the yeast ubiquitin-mediated endocytic pathway.