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J Biol Chem, Vol. 273, Issue 51, 34454-34462, December 18, 1998
From the Biology Department, Queens College and the Graduate School
of the City University of New York, Flushing, New York 11367
In Saccharomyces, the addition of
glucose induces a rapid degradation of maltose permease that is
dependent on endocytosis and vacuolar proteolysis (Medintz, I., Jiang,
H., Han, E. K., Cui, W., and Michels, C. A. (1996) J. Bacteriol. 178, 2245-2254). Here we report on the role of
ubiquitin conjugation in this process. Deletion of DOA4,
which causes decreased levels of available ubiquitin, severely
decreases the rate of glucose-induced proteolysis, and this is
suppressed by the overproduction of ubiquitin. Overexpression of
ubiquitin in an endocytosis-deficient end3-ts strain
results in the glucose-stimulated accumulation of a larger molecular
weight species of maltose permease, which we demonstrate is a
ubiquitin-modified form of the protein by utilizing two ubiquitin
alleles with different molecular weights. The size of this
ubiquitinated species of maltose permease is consistent with
monoubiquitination. A promoter mutation that reduces expression of
RSP5/NPI1, a postulated ubiquitin-protein ligase, dramatically reduces the rate of glucose-induced proteolysis of
maltose permease. The role of various ubiquitin-conjugating enzymes was
investigated using strains carrying mutant alleles ubc1 Glucose regulates maltose transport in Saccharomyces at
several levels. It blocks transcription of the maltose permease gene by
multiple mechanisms cumulatively referred to as glucose repression (2),
and it inactivates maltose permease by a process referred to as
glucose-induced inactivation or catabolite inactivation (1, 3).
Together, these processes allow for the rapid shift from maltose to
glucose fermentation. Previously, we showed that glucose-induced
inactivation of maltose permease consists of two apparently independent
processes: the proteolysis of maltose permease protein and the rapid
inhibition of maltose transport activity, which occurs even faster than
can be explained by loss of the protein alone (1). Molecular genetic
analysis using mutations in END3, REN1/VPS2,
PEP4, and PRE1 and PRE2 demonstrated
that the proteolysis of maltose permease is dependent on endocytosis, vesicle sorting, and vacuolar proteolysis and is independent of the proteasome.
Studies of a variety of different nutrient transporters suggest that
the inactivation and/or degradation of permeases is a generalized
mechanism used to respond to changes in nutrient availability from less
desirable nutrient sources or starvation conditions to preferred
nutrients and rich medium. The general amino acid permease Gap1 protein
is inactivated by the addition of ammonium ions to yeast cells growing
on proline as the sole nitrogen source (5, 6). Inactivation occurs as a
2-fold process with enzymatic inactivation by phosphorylation preceding
degradation of the permease (5, 6). The high affinity Pho84 phosphate
transporter undergoes rapid degradation once the supply of phosphate
and/or carbon source is exhausted (7). Uracil permease (Fur4p) is
phosphorylated on serine residues at the plasma membrane and is rapidly
degraded under adverse growth conditions (8). A common feature of the degradation of the maltose, galactose, uracil, and general amino acid
permeases is that all are mediated by endocytosis and subsequent transport to the vacuole, the site of degradation. Ubiquitination has
been implicated as the mechanism marking these proteins and several
others for rapid endocytosis and selective degradation (4, 6,
8-11). We report here that ubiquitination of the maltose permease
occurs in response to glucose and explore the cellular components
involved in this process.
Ubiquitination of Saccharomyces Ste2 protein, Additional studies also have implicated ubiquitination as a signal for
the endocytosis and vacuolar degradation of other plasma membrane
proteins including mammalian peptide hormone receptors (reviewed in
Ref. 13). The yeast ABC transporter Ste6 accumulates in a ubiquitinated
form in the plasma membrane of strains that are deficient in
endocytosis (11). In strains that have normal endocytotic functions,
this protein is generally found associated with internal membranes.
Another protein from this same family of yeast transporters, the
multidrug transporter Pdr5, also is ubiquitinated prior to endocytosis
and degradation in the vacuole, suggesting that ubiquitination may
trigger the endocytosis of this short lived protein (14). Similar
results have been reported for the human fibroblast growth factor
receptor (15). Moreover, many other plasma membrane receptor proteins
are found as ubiquitin conjugates including the lymphocyte homing
receptor, the platelet-derived growth factor receptor, the c-Kit
receptor, and the mammalian immunoglobulin E receptor (16-19).
In this study, we used molecular genetic analysis to explore the role
of ubiquitin in the glucose-induced inactivation of the maltose
permease. Our results indicate that loss of free ubiquitin, via a
DOA4 null mutation, impairs the glucose-induced proteolysis of maltose permease and that the effects of the doa4 Strains and Plasmids--
The Saccharomyces
cerevisiae strains used in this study and their relevant genotypes
are listed in Table I. Plasmid pDOA4-8 carries the wild-type allele of DOA4. Plasmid YEp96
(pCUP1-Ub) contains UBI4 encoding ubiquitin expressed from
the copper-inducible CUP1 promoter, and YEp105 (pCUP1-mycUb)
contains a c-myc-tagged ubiquitin allele also expressed from
the CUP1 promoter (20, 21). These plasmids were obtained
from Mark Hochstrasser (University of Chicago). Plasmid pUN70 serves as
a yCP vector control (22) as does plasmid yATAG200 (pCUP1-vector),
which contains a CUP1 promoter without any fused gene
sequence.
Plasmids pRS416-MAL61/HA, pUN70-MAL61/HA, pRS415-MAL61/HA, and
pUN30-MAL61/HA all carry the HA-tagged maltose permease under the
control of its native promoter (22, 23). Plasmids pUN90-MAL63, pUN30-MAL63, and YCP50-MAL63 all carry the MAL63 MAL-
activator gene, required in many strains for maltose-induced expression of the MAL structural genes.
Plasmid pADH1-MAL61 expressing the MAL61/HA gene from the
constitutive ADH1 promoter was constructed as follows. Using
in vitro mutagenesis, an XhoI site was introduced
into pUN30-MAL61/HA 12 base pairs upstream of the start codon
of the permease gene MAL61/HA. The promoter
sequence of this gene was removed by digestion with
XhoI and SacI and replaced with the 400-base pair
ADH1 promoter, amplified from plasmid pGAD424
(CLONTECH Inc., Palo Alto, CA) by polymerase chain reaction.
Strain Construction--
Strain CMY1025 is a maltose fermenting
leucine+ haploid segregant from a diploid obtained by
mating strains CMY1001 and PMY270, which carries a
doa4 Inactivation Assay--
The standard inactivation assay protocol
was used as described previously (1). Unless otherwise indicated, cells
were grown at 30 °C to early log phase (A600
0.1-0.3) in YP (rich) or SM (selection) medium containing 2% maltose,
harvested by filtration with cellulose filters, and resuspended in
nitrogen starvation medium (1.74 g/liter of yeast nitrogen base without
amino acids and ammonium sulfate) plus 2% (w/v) carbon source, usually
glucose. At selected time intervals, cells were harvested by filtration for Western analysis and maltose transport assays. All values depicted
in this study are the average of at least two experiments and were
carried out in duplicate. Variation was less than 15%. Growth dilution
was calculated as the A600 at time 0 divided by the A600 at time x.
Maltose Transport Assay and Maltase Assay--
Maltose
transport was measured by the uptake of 1 mM
[14C]maltose as described previously (1, 26). Transport
assays were done in duplicate on at least duplicate cultures. Maltase
activity was determined as described previously (27). Maltase activity describes the nmol of p-nitrophenol
Western Analysis and Quantitation of Relative Protein
Levels--
Cells were harvested, and total protein extracts were
prepared by the methods described previously (1, 28). Equal amounts of
total protein are loaded per well for comparison of time courses or
relative protein levels. SDS-polyacrylamide gel electrophoresis analysis and detection were carried out for the HA-tagged Mal61 maltose
permease (1). The intensity of the signal was quantitated by scanning
films with a Beckman DU640 spectrophotometer, and relative Mal61/HA
protein levels were determined by comparison of the area under the
curve. Western blots were done in duplicate on all samples for
duplicate experimental cultures, and densitometer quantitation of the
relative protein levels was carried out twice for each sample lane
(1).
Ubiquitin Is Required for Glucose-induced Proteolysis of Maltose
Permease--
The yeast DOA4 gene encodes a ubiquitin
hydrolase enzyme that functions late in the proteasomal degradation
pathway by cleaving and recycling ubiquitin from substrate remnants
still bound to protease (24). Although Doa4p is only one of several
species of ubiquitin hydrolase enzymes found in
Saccharomyces, loss of the DOA4 gene product
significantly decreases the rate of ubiquitin recycling and severely
decreases levels of available ubiquitin. We used a doa4
Glucose-induced inactivation of maltose permease was characterized in
the doa4
In order to test the possibility that the ubiquitin deficiency in the
doa4 Maltose Permease Is Ubiquitinated in Response to Glucose--
In
order to determine whether maltose permease is ubiquitinated directly,
we used strain CMY1004, which contains a temperature-sensitive allele
of END3 to slow down endocytosis and degradation of maltose permease and thereby enhance the levels of any putative ubiquitinated species (1). END3 is an early function in the endocytosis
process (30). We have shown that endocytosis and the subsequent
proteolysis of Mal61/HA maltose permease are completely inhibited at
the nonpermissive temperature, in end3-ts strains, and that,
even at the permissive temperature, maltose permease protein
accumulates to higher levels in the plasma membrane (1). Plasmid
pCUP1-mycUb (yEP105), which encodes a c-myc-tagged allele of
ubiquitin, was introduced into strain CMY1004.
Strain CMY1004 (pCUP1-mycUb) was grown to very early log phase
(A600 0.2-0.3) in selective media plus 2%
maltose at room temperature, and 0.1 mM CuSO4
was added to induce expression of c-myc-Ub (29). After a 5-h ubiquitin induction period, the culture was transferred to
37 °C to inhibit endocytosis, and then after an additional 1 h
glucose was added to a final concentration of 2%. Samples were
collected at time points throughout this process, and the level of
Mal61/HA protein was determined by Western blotting. Published reports
indicate that substrate proteins that are conjugated with a
c-myc-tagged allele of ubiquitin are more stable than
untagged ubiquitin-substrate protein conjugates and accumulate to a
significantly higher level (21).
As is evident in Fig. 2, overexpression
of ubiquitin during the Cu2+ induction period results in
the accumulation of a larger molecular weight species of Mal61/HA
protein in the end3 strain even prior to the addition of
glucose. Thus, it appears that a small amount of a putative
ubiquitinated species of maltose permease is present during growth on
maltose. The addition of the 2% glucose to the growth medium causes an
increase in the abundance of this larger molecular weight band, which
peaks at about 30-45 min.
In order to confirm that this higher molecular weight species is indeed
a ubiquitinated maltose permease, we utilized the modest molecular
weight difference produced by conjugation to c-myc-tagged
ubiquitin versus untagged ubiquitin. The difference in size
between the product encoded by these two alleles, approximately 1.3-1.5 kDa, previously has been used to verify ubiquitinated substrates such as the Mat
Strain CMY1004 (pCUP1-mycUb) expressing the Cu2+-inducible
c-myc-tagged ubiquitin and strain CMY1004 (pCUP1-Ub)
expressing the Cu2+-inducible untagged ubiquitin were both
grown at room temperature to early log phase, and 0.1 mM
CuSO4 was added to the growth media. After 4 h, the
cultures were moved to 37 °C for 1 h prior to the addition of
2% glucose. After the glucose was added, cells were allowed to
continue growing at 37 °C for 1/2 h and then harvested for
Western analysis of Mal61/HAp.
As is seen in Fig. 3, both strains
carrying the different alleles of ubiquitin exhibit the higher
molecular weight species of Mal61/HA protein described above, but in
the strain carrying the c-myc-tagged allele of ubiquitin,
this species is slightly larger than the corresponding species in the
strain carrying the untagged allele of ubiquitin. The
c-myc-tagged ubiquitin-maltose permease conjugate also
appears to be significantly more abundant than the corresponding
untagged species, consistent with reports that the c-myc
ubiquitin-conjugated proteins are more stable (21). These results
confirm that this higher molecular weight species is indeed a
ubiquitinated maltose permease. The size of this ubiquitinated species
of maltose permease is increased by approximately 6-7 kDa, compared
with the major species of Mal61/HAp, and is consistent with a
monoubiquitinated maltose permease.
RSP5/NPI1 Plays a Role in the Glucose-induced Proteolysis of
Maltose Permease--
RSP5/NPI1 encodes a ubiquitin-protein
ligase that participates in the induced degradation of at least two
permeases: the general amino acid permease, encoded by GAP1,
and the uracil permease, encoded by FUR4 (6). An
rsp5/npi1 mutant allele was isolated based on its
nitrogen repression-resistant phenotype (31) and has since been shown
to be a Ty1 insertion into the RSP5 promoter (6). Strains
carrying this mutant allele synthesize significantly reduced levels of
this essential protein that are adequate for cell growth but
insufficient for ammonium ion-induced proteolysis of Gap1 permease
(32).
To characterize the role of RSP5/NPI1 in glucose-induced
proteolysis of maltose permease, a plasmid-borne epitope-tagged maltose permease gene, MAL61/HA, was introduced into isogenic
RSP5/NPI1 and rsp5/npi1 strains. The results of
inactivation assays carried out on these two strains are shown in Fig.
4. Rapid glucose-induced proteolysis of
the Mal61/HA maltose permease is seen in the RSP5/NPI1 strain, but this rate is dramatically reduced (approximately
5-10-fold) in the rsp5/npi1 mutant strain. As Table II
shows, the rsp5/npi1 strain also expresses slightly higher
maltose transport activity in maltose-grown cells, which is consistent
with a decrease in rapid maltose permease turnover. These results
indicate that RSP5/NPI1 plays an important role in the
glucose-induced proteolysis of maltose permease.
Role of E2 Encoded by UBC1, UBC4, and UBC5 in Glucose-induced
Inactivation of Maltose Permease--
The E2 enzymes catalyze the
covalent attachment of ubiquitin to substrate proteins. At least 13 UBC genes have been identified in yeast, and they function
in many diverse aspects of cellular biology including DNA repair, cell
cycle, protein degradation, and peroxisome biogenesis (reviewed in
Refs. 13 and 33). UBC1, UBC4, and UBC5 are
implicated in the bulk degradation of short lived and abnormal proteins
and are implicated in the degradation of the transcription factor
Mat
A series of isogenic ubc mutant strains, carrying
ubc1
Table III compares the level of maltose
transport activity, maltase activity, relative maltose permease protein
levels, and maltose fermentation in these maltose-grown ubc
mutant strains transformed with MAL61/HA (columns 3-6). The
single mutant strains exhibit between 20% (ubc1
To investigate this further, Mal61/HAp was expressed in this same
series of strains except from the constitutive ADH1 promoter (results in Table III, column 7). Again, reduced levels of Mal61/HA protein are detected in all of the mutant strains but particularly in
the ubc1
In an effort to determine the rate of glucose-induced proteolysis of
maltose permease in the ubc1 CDC34/UBC3 and UBC9 Do Not Function in the Glucose-induced
Proteolysis of Maltose Permease--
Jiang et al. (35)
identified two glucose-sensing/signaling pathways that stimulate
glucose-induced inactivation of maltose permease. Pathway 1 transmits a
Rgt2p-dependent glucose signal and utilizes Grr1p as a
downstream component. Saccharomyces Grr1p is an F-box
protein (36). F-box proteins are substrate-specific adaptor proteins
that recruit various substrates to a core ubiquitination complex
referred to as the SCF complex because of the presence in the complex
of Sk1p, Cdc53p, and the F-box protein (36). SCF complexes, along with
particular Ubc enzymes, participate in the coordination of many
cellular processes through targeted degradation of specific proteins.
The yeast CDC34 (UBC3) gene encodes an essential
ubiquitin-conjugating enzyme, and is found in the Cdc4p-containing SCF
complex required for Sic1p degradation and G1/S transition,
DNA replication, and spindle pole body separation (37-41).
The Ubc enzyme that functions with the Grr1p-containing SCF complex has
not been identified (42). Given the involvement of Grr1p in pathway 1, we wished to test the possibility that Cdc34p also is involved in
glucose-induced inactivation of maltose permease. For this purpose, a
strain carrying the cdc34-2ts mutant allele (37) was
transformed with plasmids carrying the MAL63 MAL-activator
and MAL61/HA. Cells were grown in selective medium plus 2%
maltose to early log phase at the permissive temperature, 23 °C, at
which time the temperature was raised to 37 °C for 2 h prior to
the start of the inactivation assay, which was carried out at 37 °C.
The control culture was maintained at 23 °C throughout the
experiment. No significant effect is observed on the kinetics of
maltose permease inactivation and proteolysis (Fig.
6). Maltose transport activity of this
strain grown at the permissive temperature is 4.39 nmol/mg (dry
weight)/min and is essentially unchanged (4.43 nmol/mg (dry
weight)/min) after 2 h at the nonpermissive temperature.
Recent evidence indicates that the ubiquitin-like protein Smt3p of
Saccharomyces and SUMO-1, its mammalian homolog, are
covalently attached to other proteins posttranslationally (43).
UBC9, an essential yeast gene, is required for Smt3
conjugation in vivo (43). This suggests that UBC9
functions as an E2-like protein in a Smt3p conjugation pathway
analogous to ubiquitin-conjugating enzymes. Tir1p, an Arabidopsis
thaliana F-box-containing homologue of Grr1p is a downstream
component in the jasmonate-sensing pathway and functions in the
conjugation of Rub1p, another ubiquitin-like homologue, to target
proteins (44). For these reasons, we decided to test the possibility
that Ubc9p is involved in glucose-induced proteolysis of maltose permease.
Isogenic UBC9 and ubc9-ts strains were
transformed with plasmids containing the MAL63 MAL-activator
and MAL61/HA genes (45). The resulting transformants were
grown to early log phase at room temperature in selective medium plus
2% maltose and equilibrated at 37 °C for 2 h. Standard
inactivation assays were carried out at the nonpermissive temperature
of 37 °C. The results shown in Fig. 6 demonstrate that loss of Ubc9p
function has no significant effect on glucose-induced inactivation of
maltose permease. The rate of loss of maltose transport activity and
proteolysis of maltose permease protein are comparable in both strains.
Steady state maltose transport activity following assays at 37 °C in both strains also was comparable at 2.82 and 2.81 nmol/mg (dry weight)/min for the UBC9 and ubc9-ts strains, respectively.
The results described above strongly suggest that ubiquitination
of maltose permease is an essential early step in the rapid glucose-induced proteolysis of maltose permease. Several lines of
evidence support this conclusion. First, by utilizing a
c-myc-tagged allele of ubiquitin that produces more stable
and more abundant protein-conjugates than its untagged ubiquitin
counterpart (21), we demonstrated the accumulation of a higher
molecular weight species of MAL61/HAp in strains that express this
tagged ubiquitin allele (Fig. 2). The higher molecular weight species
of MAL61/HAp is a ubiquitin-conjugated maltose permease based on a size
shift observed when different molecular weight ubiquitin alleles were utilized (Fig. 2). This technique has been used previously to demonstrate ubiquitin conjugates of the Mat Second, the rate of glucose-induced proteolysis of Mal61p is slowed in
a doa4 Third, reduced levels of Rsp5p/Npi1p dramatically decrease the rapid
rate of glucose-induced proteolysis of maltose permease (Fig. 4). The
ubiquitin-protein ligase encoded by RSP5/NPI1 has previously
been shown to be necessary for the induced degradation of the general
amino acid and uracil permeases (6, 8) as well as for the
internalization of Ste2p.2
RSP5/NPI1 may be associated with the membrane at certain
times (6), a fact that is consistent with its role in ubiquitination of
maltose permease at the plasma membrane.
The following findings are also consistent with the conclusion that
ubiquitination of maltose permease marks this protein for degradation.
Ubiquitin-conjugated maltose permease appears to be in relatively low
abundance compared with the level of unubiquitinated maltose permease.
This is similar to results demonstrated for the The ubiquitin-conjugated maltose permease species observed in Figs. 2
and 3 has an apparent molecular mass approximately 7-8 kDa higher than
the nonubiquitinated maltose permease when viewed on SDS-polyacrylamide
gels. This is likely to correspond to a monoubiquitinated form. Studies
of a truncated allele of Ste2p demonstrated a ligand-induced
monoubiquitination sufficient for internalization and vacuolar
degradation (10, 46). These authors suggest that a single ubiquitin
moiety, as opposed to the polyubiquitin chains preferred by the
proteasome, is recognized by the endocytotic machinery. Studies
using the Lys to Arg mutant alleles of ubiquitin unable to form
polyubiquitin chains do not indicate a decreased efficiency of
degradation (46). This is in contrast with results reported for the
uracil permease by Galan and Haguenauer-Tsapis (47), who found that
mutations at Lys-29 and Lys-48 had no effect on induced turnover but,
when polyubiquitination was blocked at Lys-63, the rate of Fur4p
endocytosis was reduced but not eliminated.
Experiments are under way to identify the site(s) of ubiquitination in
Mal61p. Mutation of the lysine residue in the DAKISS sequence of the
truncated Ste2p or on all seven lysine residues of the C-terminal
cytoplasmic domain block endocytosis, clearly demonstrating a direct
requirement for ubiquitin in Ste2p endocytosis (10, 46). Mutation of a
"destruction box" sequence in Fur4p stabilized that permease
against stress-induced degradation (48). Moreover, mutation of a single
lysine to alanine (R294A) is resistant to stress-induced degradation
(8).
The UBC1, UBC4, and UBC5 genes have
been implicated as the E2 enzymes required for substrate conjugation of
certain yeast plasma membrane proteins including the Our results add the Mal61/HA maltose permease to the growing list of
permeases, receptors, and other plasma membrane proteins that are
marked by ubiquitination for rapid endocytosis and lysosomal (vacuolar)
degradation. Moreover, for maltose permease and all of the other yeast
membrane proteins for which this has been studied, Npi1p/Rsp5p appears
to play an important role. Npi1p/Rsp5p contains a so-called
C2 domain also found in certain isoforms of protein kinase
C and believed to mediate phospholipid interaction (6). This makes
Npi1p an interesting candidate for a membrane-associated ubiquitin-protein ligase. Studies are currently under way to identify the target sites of this ubiquitination in maltose permease. The target
sites in both Ste2p and Fur4p have been identified, and both appear to
require phosphorylation of key serine residues prior to ubiquitination.
Our published results demonstrated that Mal61/HAp is phosphorylated and
that the level of phosphorylation is increased by glucose (1).
Therefore, we are also exploring the role of phosphorylation in the
glucose-induced proteolysis of maltose permease and are attempting to
identify the kinase(s) involved.
We thank Mark Hochstrasser and Phoebe Johnson
for critical discussions and the gift of strains and plasmids. We also
thank Pat McGraw and Bruno Andre for the gift of strains and plasmids. We thank Thomas Hradeck, Susan Rotenberg, and Xiao-Guang Sun for technical advice and assistance.
*
This work was supported by NIGMS, National Institutes of
Health, Grant GM49280 (to C. A. M.).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.
§
To whom correspondence should be addressed: Biology Dept., Queens
College, CUNY, 65-30 Kissena Blvd., Flushing, NY 11367. Tel.:
718-997-3410; Fax: 718-997-3445; E-mail: corinne_michels{at}qc.edu.
The abbreviations used are:
E2, ubiquitin
carrier protein; HA, hemagglutinin.
2
R. Dunn and L. Hicke, personal communication.
3
R. Haguenauer-Tsapis, unpublished results.
The Role of Ubiquitin Conjugation in Glucose-induced Proteolysis
of Saccharomyces Maltose Permease*
,, and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
ubc4, ubc4 ubc5,
cdc34-ts2/ubc3, and ubc9-ts. Loss
of these functions was not shown to effect glucose-induced proteolysis of maltose permease, but loss of Ubc1, -4, and -5 was found to inhibit
maltose permease expression at the post-transcriptional level.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-factor
receptor, is required for its ligand-stimulated endocytosis and
vacuolar proteolysis (10). END4 mutations inhibit
endocytosis of -factor and stimulate the appearance of
multiubiquitinated species. A sequence in the C-terminal cytoplasmic
domain of Ste2p, SINNDAKSS (12), is sufficient to stimulate
endocytosis, but mutation of the Lys in this target sequence to Arg
inhibits ligand-stimulated ubiquitination and degradation. These
results clearly implicate ubiquitination in receptor targeting to
endocytosis. Ubiquitination also is required for endocytosis of yeast
uracil permease (8) and probably the galactose transporter, Gal2p
(4).
null
mutation can be suppressed by the overexpression of ubiquitin. We
demonstrate that the maltose permease exists as a ubiquitinated species
and that the amount of this ubiquitinated species increases
dramatically upon the addition of glucose to maltose fermenting cells.
Rsp5/Npi1 ubiquitin-protein ligase is implicated in the proteolysis of
maltose permease. Mutations in UBC1, UBC4, and
UBC5 encoding ubiquitin conjugation enzymes UBC (ubiquitin
carrier proteins; E21
enzymes), in combination were found to dramatically decrease the level
of maltose permease expressed, apparently by affecting a
post-transcriptional process but not glucose-induced proteolysis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
S. cerevisiae strains used in this study
::LEU2 disruption (24). Southern analysis using MAL61-specific probes revealed the presence two
maltose permease genes, one at the MAL1 locus
(MAL61/HA, derived from CMY1001) and a second
(MAL31, derived from PMY270) mapping to the partially
functional MAL3 locus encoding MAL31 (maltose
permease) and MAL32 (maltase) (25).
-D-glucopyranoside cleaved per mg of protein per min as
measured spectrophotochemically.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
null mutant strain to explore the dependence on ubiquitin of
glucose-induced proteolyis of maltose permease.
null strain, CMY1025, and as a control in strain
CMY1025 carrying the wild-type DOA4 gene on a CEN
plasmid. As is evident from Fig. 1
(top two panels), the doa4
mutant strain exhibits a dramatically decreased rate of glucose-induced
proteolysis of Mal61/HA permease, to the extent that the loss of
maltose permease protein parallels the growth of the culture (growth
dilution). In comparison, in the DOA4 strain, maltose
permease protein is degraded more rapidly than can be expected from
growth alone. Table II indicates that the
steady state rate of maltose transport in the doa4 strain
is slightly higher (37%) than that of a strain expressing the
wild-type DOA4 gene, which is consistent with the decrease
in maltose permease turnover. Interestingly, despite the apparent lack
of glucose-induced proteolysis of maltose permease in the
doa4 strain, glucose stimulates a decrease in maltose transport activity, indicating that the inhibition of transport activity occurs by a process that is independent of ubiquitin availability.
View larger version (25K):
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Fig. 1.
Effects of a doa4 null
mutation on glucose-induced inactivation of maltose permease.
Strain CMY1025 (doa4) was transformed with plasmid pDOA4
carrying the DOA4 gene or plasmid pUN70 as a vector control.
Transformants were grown in selective medium plus 2% maltose and
harvested, and the standard inactivation assay was performed (as
described under "Materials and Methods"). Plasmid pCUP1-Ub (yEP96),
carrying a c-myc-tagged allele of UBI4 expressed
from the copper-inducible CUP1 promoter (21) or the vector
control plasmid pCUP1-vector (yATAG200) lacking the UBI4
insert were introduced into strain CMY1025. Transformants were grown to
early log phase in selective medium plus 2% maltose, incubated for
4 h with 0.1 mM copper sulfate, harvested, and
transferred to nitrogen starvation medium plus 2% glucose. At the
indicated times, the A600 was determined, and
aliquots of culture were removed for maltose transport assays and the
preparation of total protein extracts for Western analysis of Mal61/HA
protein levels as described under "Materials and Methods."
Representative Western blots are shown, but the quantitation data used
in the graph was obtained from the average of at least two experimental
cultures with samples each run on duplicate gels, and scanned twice.
The relative levels of Mal61/HA protein (
) and maltose permease
transport activity (
) compared with the zero time sample are plotted
along with the growth dilution (
). Growth dilution represents the
growth of the culture during the course of the experiment and is
calculated as the A600 at time 0 divided by the
A600 at time x.
Maltose transport rates of doa4 and rsp5/npi1 mutant
strains
strain is responsible for the decreased rate of glucose-induced proteolysis of maltose permease, we determined whether
overexpression of ubiquitin could overcome the loss of active ubiquitin
recycling. Plasmid yEP96 (pCUP1-Ub), carrying the ubiquitin gene
UBI4 fused to the copper-responsive promoter from
CUP1, was introduced into the doa4 mutant
strain CMY1025 (20). The standard inactivation assay was carried out,
except 0.1 mM copper sulfate was added to the culture
medium 4 h prior to the transfer to glucose and the initiation of
the inactivation assay (29). As can be seen in Fig. 1
(bottom two panels), overexpression of
ubiquitin in the doa4 (pCUP1-Ub) strain suppresses the
loss of DOA4, restoring a more rapid rate of glucose-induced
proteolysis of maltose permease than that observed in the
doa4 (pCUP1-vector) control strain. Table II shows that
the steady state transport rate of maltose in a
ubiquitin-overexpressing doa4 strain, CMY1025 (pCUP1-Ub),
is half that seen in the control strain, CMY1025 (pCUP1-vector). This
is also consistent with the proposal that ubiquitin is required for
rapid turnover of maltose permease.
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Fig. 2.
Overexpression of a c-myc-tagged
ubiquitin allele in an end3-ts strain. Strain CMY1004,
containing an end3-ts allele, was transformed with plasmid
pCUP promoter-mycUb (yEP105) encoding a c-myc-tagged allele
of ubiquitin on a copper-inducible promoter, CUP1. The
resulting strain was grown to early log phase at room temperature and
then incubated with 0.1 mM copper sulfate for 5 h. At
the 4th h of incubation, the medium was moved to 37 °C to inhibit
endocytosis. After a total of 5 h of incubation, 2% glucose was
added to the growth medium. At the selected time points, aliquots were
collected for Western analysis as described under "Materials and
Methods." The lane labeled CMY1004 depicts
Western analysis of that strain grown in maltose lacking the
pCUP1-mycUb (yEP105) plasmid.
2 transcriptional regulator (8, 9, 14,
21).
View larger version (38K):
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Fig. 3.
Western blot analysis of an end3-ts
strain expressing two different molecular weight alleles of
ubiquitin. Strain CMY1004 was transformed with plasmid pCUP1-Ub
(yEP96) carrying UBI4 expressed from the CUP1
promoter or pCUP1-mycUb (yEP105) encoding a c-myc-tagged
allele of ubiquitin also expressed from the CUP1 promoter.
Both strains were grown to early log phase at 30 °C in selective
media plus 2% maltose and then incubated in 0.1 mM copper
sulfate for 4 h, the last hour at 37 °C to inhibit endocytosis.
2% glucose was added for 1/2 h prior to harvesting the cells
for Western analysis of Mal61/HA protein.
View larger version (22K):
[in a new window]
Fig. 4.
Effects of reduced expression of Rsp5/Npi1
ubiquitin-protein ligase on glucose-induced inactivation of maltose
permease. Isogenic RSP5/NPI1 and rsp5/npi1
strains were transformed with a plasmid expressing the
Mal61/HA gene. A strain carrying the npi1
mutation isolated by Grenson (31) was utilized that expresses
significantly reduced levels of Rsp5/Npi1 protein (32). The standard
inactivation assay protocol was carried out. The relative levels of
Mal61/HA protein ( ) and maltose transport activity () compared
with the zero time sample are plotted along with the growth dilution
() as described in the legend to Fig. 1.
2, the -factor receptor Ste2p, and the a-factor
receptor Ste3p (9, 10, 13, 33). UBC6 and UBC7
also function in the degradation of the Mat2 repressor through a
pathway that is distinct from that of UBC4 and
UBC5 (34). The functions of several E2 enzymes, like Ubc4/5
and Ubc6/7, overlap, since the most dramatic results are seen only in
double mutants. Ubiquitination, in some cases via the Ubc4/5 E2
enzymes, has been implicated in the signaling of endocytosis and
degradation of many yeast membrane proteins including Ste2p, Gap1p,
Fur4p, Ste3p, and Pdr5p (6, 9, 10, 11, 14, 33). We explored the role of
these E2 enzymes in glucose-induced inactivation of maltose permease.
, ubc4, or ubc5 alleles, were
transformed with CEN plasmids carrying the
MAL-activator gene MAL63 and the HA-tagged
maltose permease gene MAL61/HA, and the half-life of maltose
permease was determined using the standard inactivation assay. The
single mutant strains showed no significant change in half-life of the
permease compared with the parental strain: UBC1 UBC4 UBC5
(O.4 h), ubc1 (0.5 h), ubc4 (0.5 h), and
ubc5 (0.3 h). Rather unexpectedly, double mutant strains
containing either the ubc1 ubc4 or ubc4
ubc5 double null mutation expressed no detectable maltose
permease protein, suggesting a possible role for these gene functions
in maltose permease expression either at the transcription or
post-translational level.
) and
35% (ubc5) of the level of maltose permease protein
expressed by the UBC1,4,5 parental strain, and these levels
also are paralleled by a decrease in maltose transport activity. All
single mutant strains fermented maltose in 1 day. The ubc1
ubc4 and ubc4 ubc5 double mutant strains
expressed only 5-10% of the parental levels of maltose transport
activity and took 7-9 days to ferment maltose. Interestingly, maltase
activity in these strains does not correlate with the levels of maltose permease. The maltase gene is divergently transcribed from a shared promoter with the maltose permease gene, and both genes are
coordinately regulated. No significant variation from the parental
strain is seen in single deletion mutant strains, and maltase levels
are actually increased in the ubc1 ubc4 double mutant
about 2-fold above that of the parental strain. Only in the
ubc4 ubc5 strain were the levels of maltase
significantly decreased, but not to the same extent of maltose
transport levels. Expression from MAL62 promoter-LacZ (MAL62 encodes maltase) and
MAL61 promoter-LacZ reporter constructs also were
tested in these ubc strains in order to monitor the effects
of these mutations on transcription initiation. Results (data not
shown) were consistent with the expression levels of maltase,
suggesting that the effect of loss of Ubc1/4 or Ubc4/5 ubiquitin-conjugating enzymes is not at the level of transcription initiation.
Effect of ubc1, ubc4, and ubc5 mutations on MAL gene expression
, ubc1 ubc4, and ubc4 ubc5
strains. Thus, these results also suggest a role for Ubc1, -4, and -5 in a post-translational process required for maltose permease expression.
ubc4 and ubc4
ubc5 double mutant strains, we used the strains that express
MAL61/HA from the ADH1 promoter. These strains,
grown on glycerol/lactate as the carbon source, accumulate levels of
maltose permease adequate (approximately 25% of wild type; data not
shown) to allow us to carry out an inactivation assay. The parental
strain and the ubc1 ubc4 and ubc4
ubc5 double mutant strains carrying plasmid pADH1-MAL61/HA were
grown to early log phase in selective medium plus 3% glycerol, 2%
lactate. A standard inactivation assay was carried out with the
exception that 12.5 µg/ml cyclohexamide was added to the 2% glucose
inactivation medium at time 0 to stop the continued synthesis of
maltose permease. The results shown in Fig.
5 do not demonstrate a significant effect
on the rate of proteolysis in the mutant strains. The half-life in both
the parental strain and the ubc1 ubc4 strain is about
0.5 h, and in the ubc4 ubc5 double mutant it is
only increased about 2-fold.
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Fig. 5.
Effects of glucose-induced inactivation on
ubc1,4 and ubc4,5 strains that express
MAL61/HA on a constitutive ADH1 promoter.
Strains were grown in selective media plus 3% glycerol, 2% lactate to
early log phase. After harvesting, cells were resuspended in
inactivation media supplemented with 12 µg/ml of cyclohexamide, and
the standard inactivation assay was carried out as described in the
legend to Fig. 1 and under "Materials and Methods." The relative
levels of Mal61/HA protein () and maltose transport activity ()
compared with the zero time sample are plotted along with the growth
dilution ().
View larger version (25K):
[in a new window]
Fig. 6.
Glucose-induced inactivation of MAL61/HAp in
cdc34-ts, and ubc9-ts strains. Strain
MGG15 containing the cdc34-2ts allele was transformed
with plasmids containing the MAL61/HA gene and the
MAL activator gene. Following growth to early log phase at
room temperature, the standard inactivation assay protocol was carried
out at the permissive temperature, 23 °C, and at the nonpermissive
temperature 37 °C. Strains FM394, UBC9, and FM395,
ubc9-ts, were transformed with plasmids containing the
MAL/61HA allele and the MAL63 MAL activator
genes. Strains were grown to early log phase in maltose media at
23 °C, equilibrated to 37 °C for 2 h, and then transferred
to nitrogen starvation media plus 2% glucose at 37 °C. The standard
inactivation assay was carried out at 37 °C, as described in the
legend to Fig. 1 and under "Materials and Methods." The relative
levels of Mal61/HA protein ( ) and maltose transport activity ()
compared with the zero time sample are plotted along with the growth
dilution ().
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
2 transcriptional activator, the ABC transporter Ste6, and the Pdr5 multidrug transporter (11, 14, 21). We also show that the abundance of this ubiquitinated species of Mal61/HAp dramatically increases upon the addition of
glucose to the growth medium (Fig. 3).
strain (Fig. 1). DOA4/UBP4 encodes a
ubiquitin-hydrolase that is localized to the 26 S proteasome and
appears to play an important role in maintenance of free unconjugated
ubiquitin pools (33). Loss of Doa4p affects a variety of physiological
functions, suggesting decreased levels of available ubiquitin, at least
in certain compartments (24). The Doa4p deficiency in
glucose-stimulated Mal61p turnover can be overcome by overproduction of
ubiquitin (Fig. 1), indicating that the slow rate of proteolysis in
this strain is directly attributable to the depletion of available ubiquitin.
-factor receptor and
the yeast uracil permease (8, 10). Additionally, evidence exists to
support the proposal that conjugation of ubiquitin to maltose permease
takes place at the plasma membrane prior to endocytosis. The
MAL61/HAp-ubiquitin conjugate accumulates in an end3-ts
strain that is deficient for endocytosis at the nonpermissive
temperature, and this strain even accumulates MAL61/HAp at the membrane
at the permissive temperature (1).
-factor
receptor, the a-factor receptor, and the ABC transporter Ste6p (9-11).
Double mutant ubc4 ubc5 strains exhibit reduced rates of
induced turnover of these proteins. Ubc1p is essential in ubc4
ubc5 mutant strains, suggesting a possible role in endocytosis of
membrane proteins (33). We explored the role of these
ubiquitin-conjugating enzymes in maltose permease proteolysis. While we
found that Ubc1p, Ubc4p, and Ubc5p appear to play an as yet
unidentified post-transcriptional role in maltose permease expression,
our results do not support the suggestion that these proteins are
important for its glucose-induced proteolysis. This is in contrast to
results for Ste2p (10, 46) but similar to findings regarding the
inositol permease Itr1p (49) and uracil
permease.3
ACKNOWLEDGEMENTS
FOOTNOTES
This work was carried out in partial fulfillment of these
authors' requirements for the Ph.D. degree from the Graduate School of
the City University of New York.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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