(Received for publication, August 8, 1995)
From the
Catabolite inactivation of fructose-1,6-bisphosphatase (FBPase),
a key enzyme in gluconeogenesis, is due to phosphorylation and
subsequent degradation in the yeast Saccharomyces cerevisiae.
The degradation process of the enzyme had been shown to depend on the
action of the proteasome. Here we report that components of the
ubiquitin pathway target FBPase to proteolysis. Upon glucose addition
to yeast cells cultured on nonfermentable carbon sources FBPase is
ubiquitinated in vivo. A multiubiquitin chain containing
isopeptide linkages at Lys of ubiquitin is attached to
FBPase. Formation of a multiubiquitin chain is a prerequisite for the
degradation of FBPase. Catabolite degradation of FBPase is dependent on
the ubiquitin-conjugating enzymes Ubc1, Ubc4, and Ubc5. The 26 S
proteasome is involved in the degradation process.
Specific and rapid degradation of certain proteins is a
fundamental mechanism in many biological processes, including embryonic
development, cell proliferation, cell cycle control, and metabolic
regulation(1) . The pathways signaling selective proteolysis
are only very poorly understood. The regulation of FBPase, ()a key enzyme in gluconeogenesis, represents an ideal model
to study such signaling pathways. The cytoplasmic enzyme is subject to
catabolite inactivation in the yeast Saccharomyces
cerevisiae(2) . Addition of glucose to yeast cells grown
on a nonfermentable carbon source causes a rapid inactivation of FBPase
due to phosphorylation and subsequent
degradation(3, 4, 5) . Since its
discovery(6) , the proteolytic mechanism of FBPase degradation
had remained elusive. The prominent proteolytic activities of the
vacuole, especially proteinase yscB, in degrading FBPase in vitro at first suggested this lysosomal enzyme to be the catalyst of
FBPase degradation under catabolite inactivation
conditions(7) . However, experiments using mutants defective in
the activity of proteinase yscB and other vacuolar proteases seemed to
rule out this possibility(8, 9, 10) .
Recently, selective uptake of FBPase into the vacuole and degradation
by vacuolar proteases under catabolite inactivation conditions had been
proposed(11) . However, mutants defective in proteolytic
activities of the yeast proteasome revealed the involvement of this
protease complex in glucose-induced degradation of FBPase, indicating
this process to be a cytoplasmic event(12, 13) . 26 S
proteasomes are large multicatalytic protease complexes located in the
cytoplasm and nucleus of the eukaryotic cell. The 26 S proteasome is
build up from a 20 S proteasome core and two additional 19 S
subcomplexes(14, 15) . Proteasome mutants demonstrated
the involvement of the enzyme complex in stress-dependent and
ubiquitin-mediated proteolysis(16, 17, 18) . In vitro studies demonstrated that 26 S proteasomes rather
than 20 S proteasomes are able to degrade ubiquitinated proteins in an
ATP-dependent fashion(19, 20, 21) .
In
eukaryotes the ubiquitin system constitutes the major cytoplasmic
pathway targeting proteins to selective
degradation(22, 23, 24, 25, 26) .
Ubiquitin, a 76-residue polypeptide found in all eukaryotes, is
covalently attached with its carboxyl-terminal glycine to the protein
substrate through an isopeptide bond in a multistep reaction. Ubiquitin
is first activated through the formation of a thiol ester bond of the
carboxyl-terminal glycine with the ubiquitin-activating enzyme E1.
Thereafter ubiquitin is transferred to a ubiquitin-conjugating enzyme
E2, which subsequently links it to the -amino group of an internal
lysine of the substrate protein. In some cases this last step is
performed with the support of a ubiquitin ligase E3, which mediates the
recognition of the target protein together with the E2 enzyme. In
further successive reactions ubiquitin moieties are linked to the
Lys
residue of each previously conjugated ubiquitin
molecule until a polyubiquitin chain is formed(27) . The
specificity of the ubiquitin system for a certain substrate is thought
to be a property of the E2 and E3 enzymes.
In yeast, 10 UBC (E2) genes have been identified until now (23) . UBC1, UBC4, and UBC5 encode a functionally overlapping group of enzymes, which mediate bulk turnover of short-lived and abnormal proteins(28, 29) . The UBC6 gene product resides in the endoplasmic reticulum membrane and seems to be involved in degradation of endoplasmic reticulum membrane proteins (30) . UBC3 (CDC34) and UBC9 encode genes required for degradation of cyclins and regulation of the cell cycle(31, 32) . The UBC2 (RAD6) gene is involved in different processes including DNA repair and protein degradation via the N-end rule pathway(33, 34) . The UBC10 (PAS2) gene product is essential for the biogenesis of peroxisomes(35) . UBR1 is the only gene encoding an E3 enzyme that has been identified in yeast as yet(36) .
Only a few in vivo substrates have been
known up to now, which are targeted to degradation by the ubiquitin
system. They include the yeast transcriptional regulators Mat2 (37) and GCN4(38) ,
cyclins(33, 39, 40, 41) , and the
oncogene products p53 (42) and Mos (43) .
Another system rendering proteins susceptible to specific degradation via the proteasome is antizyme. Its function in the degradation of the tightly controlled ornithine decarboxylase has been shown in vitro and in vivo(44, 45) .
The signal rendering FBPase accessible to proteolysis through the proteasome during the catabolite inactivation process had remained unknown. Here we show that glucose-induced degradation of FBPase is signalled via the ubiquitin pathway.
Cells were grown in mineral medium (MV, 0.67% Difco yeast nitrogen
base without amino acids) containing 2% glucose or for derepression of
FBPase, 2% ethanol as the carbon source, and required supplements.
Radiolabeling was done in pulse medium (0.17% Difco yeast nitrogen base
without amino acid and ammonium sulfate, 0.5% proline, 100 µM ammonium sulfate, 2% ethanol, and required supplements). In all
experiments requiring induction of Ub synthesis, CuSO was
added to a final concentration of 100 µM.
Radiolabeling was done by adding
[S]methionine (Amersham, Braunschweig) to the
cell suspension to a final concentration of 45 µCi
ml
(pulse). After 1.5 h, cells were shifted onto MV
medium containing 2% glucose and 10 mM nonradioactive
methionine (chase). In the experiments overexpressing different
ubiquitin derivatives, CuSO
(100 µM) was
present during the pulse and chase periods. 1-ml samples were taken at
the times indicated, and the chase was terminated by the addition of
trichloroacetic acid (final concentration, 5%). The precipitates were
washed twice with cold ethanol (-20 °C) and dried.
Subsequently cells were lysed in 100 µl of breaking buffer (50
mM Tris, pH 7.5, 6 M urea, 1% SDS, and 1 mM
EDTA) with agitation by glass beads three times for 1 min with
intermittent heating at 95 °C, followed by a 5-min heating period
at the same temperature. The lysate was diluted with 1 ml of IP buffer
(50 mM Tris, pH 7.5, 190 mM NaCl, 6 mM EDTA,
and 1.25% Triton X-100) containing protease inhibitors (1 mM PMSF and 20 µg
ml
each of
chymostatin, pepstatin A, leupeptin, and antipain), centrifuged, and
transferred to a new test tube. FBPase antiserum (8 µl) was added,
and the samples were gently agitated for 2 h at room temperature.
Immunoprecipitates were collected by adding 50 µl of a protein
A-Sepharose Cl4-B (Pharmacia, Freiburg, Germany) suspension (5% w/v in
IP) and further incubated for 1 h. The Sepharose beads were centrifuged
and washed three times with IP buffer. Proteins were released from
Sepharose by boiling in 50-100 µl of 2 times concentrated
Laemmli sample buffer (200 mM Tris/HCl, pH 6.8, 10% SDS, 20%
glycerol, 0.2% bromphenol blue), separated by SDS-PAGE (10%), and
visualized by fluorography. Protein bands were quantitated using a Fuji
BAS 2000 bio-imaging analyzer.
Ubiquitin-mediated degradation of a protein requires the covalent attachment of ubiquitin moieties, leading to the occurrence of higher molecular mass species of such a protein. The use of an epitope-tagged ubiquitin derivative allows the unambiguous identification of such ubiquitin protein conjugates in vivo(46, 48) .
If catabolite degradation of FBPase were mediated via the ubiquitin pathway, higher molecular mass species of FBPase would be expected. When pulse-labeled FBPase was immunoprecipitated during catabolite inactivation in cells expressing wild-type ubiquitin from a 2-µm plasmid, at least two additional labeled species with higher mass than FBPase could be detected in the precipitates (Fig. 1A, lanes 2 and 3). The appearance of these larger species was only visible after shifting cells derepressed of FBPase onto glucose medium (compare Fig. 1A, lane 1 with lanes 2 and 3), suggesting that these species are intermediates of the glucose-induced degradation process of FBPase. To test whether these species actually represent ubiquitin conjugates of FBPase, a ha-tagged version of ubiquitin was overexpressed. This ubiquitin variant (haUb) is about 1.5 kDa (14 amino acids) larger than wild-type ubiquitin, resulting in a decrease of the mobility of this molecule on SDS-PAGE.
Figure 1:
FBPase is
ubiquitinated in vivo. A, pulse-chase analysis of
FBPase degradation. In addition to FBPase, FBPase immunoprecipitates
show a set of higher molecular mass species (arrows, lanes
2 and 3). Cells of W3031B overexpressing wild-type Ub
were pulse-labeled during derepression of FBPase in ethanol-containing
medium and chased with the addition of glucose, followed by extraction,
immunoprecipitation, and SDS-PAGE as described under ``Materials
and Methods.'' B, comparison of the SDS-PAGE pattern of
FBPase immunoprecipitates from labeled W3031B cells expressing either
wild-type ubiquitin (wt-Ub, lane 2) or haUb (lane
3). Expression of haUb in W3031B cells leads to an increase of the
apparent molecular masses of the putative Ub-FBPase conjugates. FBPase
null mutant (fbp1) cells expressing either wild-type Ub
or haUb show no corresponding ubiquitin conjugates (lanes 1 and 4). Samples were taken 20 min after the addition of
glucose to the cells.
If the larger protein species present in the FBPase precipitates (Fig. 1A) were ubiquitinated forms of this enzyme, in vivo substitution of wild-type ubiquitin with haUb would lead to a slower migration of these species as compared with FBPase ligated to wild-type ubiquitin.
Cells transformed with either
plasmid YEp96 expressing wild-type Ub or plasmid YEp112 expressing haUb
were pulse-labeled during derepression of FBPase. After inducing the
inactivation period by addition of glucose, samples were taken. Crude
extracts were immunoprecipitated with FBPase antibodies, and proteins
were separated by SDS-PAGE (Fig. 1B). Comparison of the
pattern of bands in lanes 2 (expression of wild-type Ub) and 3 (expression of haUb) of Fig. 1B shows that
in the presence of haUb the new protein species appearing are of higher
molecular mass than those species appearing in the presence of
wild-type Ub. This indicates that the newly appearing bands represent
ubiquitinated proteins. Moreover, the absence of the corresponding
bands in the control patterns obtained from FBPase null mutants also
expressing either wild-type or ha-tagged Ub (Fig. 1B, lanes 1 and 4) reveals that these species are
ubiquitinated forms of FBPase. To further confirm this, we tested
whether the putative FBPase-ubiquitin conjugates immunoreacted with
both FBPase and ha antibodies. Crude extracts from glucose-inactivated
cells were immunoprecipitated with FBPase antibodies; proteins were
separated by SDS-PAGE and blotted onto nitrocellulose filters. The
filters were then probed with ha antibodies. As can be seen in Fig. 2, lanes 6, 7, and 8, extracts
from cells expressing haUb show a complex spectrum of
ha-ubiquitin-FBPase conjugates after shifting cells to glucose. The
amount of ubiquitinated FBPase species is highest shortly after glucose
addition (Fig. 2, lane 6), followed by a drastic
decline along with further incubation of the cells on glucose (Fig. 2, lanes 7 and 8). This behavior is
consistent with the view that the ubiquitinated forms of FBPase are the
substrates of the degradation machinery of the cell. As expected,
neither in cells expressing wild-type Ub nor in FBPase null mutant
cells (fbp1) expressing haUb is any ubiquitin conjugate
detectable (Fig. 2, lanes 2 and 4 respectively), clearly confirming that the observed bands in Fig. 2(lanes 6-8) represent ubiquitinated forms
of FBPase. From these data we conclude that FBPase is ubiquitinated
upon glucose addition and that this event triggers catabolite
degradation of the enzyme.
Figure 2:
Immunodetection of Ub-FBPase conjugates
during catabolite inactivation. Crude extracts from cells expressing
either haUb or wild-type ubiquitin (wt-Ub) were
immunoprecipitated with FBPase antibodies and separated by SDS-PAGE.
Thereafter the proteins were transferred to a nitrocellulose membrane
and probed with ha antibodies. A variety of different haUb-FBPase
conjugates occur after shifting the cells onto glucose (lanes
6-8). The amount of these conjugates decline with increasing
time after glucose addition. The controls (FBPase null mutant (fbp1) expressing haUb and wild-type cells (FBP1) expressing wild-type Ub) display no FBPase-Ub
conjugates as expected (lanes
1-4).
It has been shown that treatment of cells with cycloheximide at the time of glucose addition prevents degradation of FBPase(11) . We found that FBPase is also not ubiquitinated under these conditions (not shown). This indicates the necessity upon catabolite inactivation of the new synthesis of a protein(s) that is part of the signaling cascade and indispensible for ubiquitination and degradation of FBPase.
For the N-end rule substrates (27) and for the Mat2 repressor (46) it has been
shown that proteolysis via the ubiquitin-mediated pathway requires
formation of a multiubiquitin chain for their efficient degradation.
The ubiquitin molecules are linked within this chain by isopeptide
bonds connecting the carboxyl-terminal Gly
of one
ubiquitin moiety to the
-amino group of Lys
of the
adjacent ubiquitin molecule. Lys
and Lys
have
also been found to be sites for polyubiquitination(49) , but
Lys
has been identified as the primary site of this
process. A modified ubiquitin carrying an arginine at position 48
(Ub-R48) instead of lysine can still be conjugated to other proteins
but fails to function as an acceptor within the multiubiquitin chain (27) . Therefore, Ub-R48 can serve as a probe for monitoring
the presence of a Lys
-linked multiubiquitin chain in a
protein of interest(6) .
Western blot analysis had indicated
the appearance of multiply ubiquitinated FBPase molecules during
catabolite inactivation (Fig. 2, lane 6). If
Lys-linked multiubiquitination is a prerequisite for
glucose-induced degradation of FBPase, the presence of Ub-R48 should
affect this process. To address this possibility we compared the
degradation rate of FBPase in cells expressing high levels of wild-type
ubiquitin (transformed with YEp96) and in cells expressing high levels
of Ub-R48 (transformed with YEp110) by pulse-chase analysis. Although
glucose-induced degradation of FBPase occurred in cells overexpressing
wild-type ubiquitin (Fig. 3A), the presence of Ub-R48
inhibited the degradation of the enzyme (Fig. 3B).
Figure 3: Overexpression of Ub-R48 inhibits glucose-induced FBPase degradation. Pulse-chase analysis in W3031B cells expressing either wild-type Ub (wt-Ub, A) or Ub-R48 (B) was done as described in the legend to Fig. 1.
It is noticeable that the expression of Ub-R48 resulted in an
increase of the amount of the monoubiquitinated FBPase species (see arrow in Fig. 3). The SDS-PAGE pattern of FBPase
immunoprecipitates from pulse-labeled cells expressing Ub-R48 did not
show multiply ubiquitinated species of FBPase (not shown). Formation of
a multiubiquitin chain containing Lys linkages seems
therefore to be necessary for glucose-induced degradation of FBPase. As
degradation of FBPase is nearly blocked in cells expressing Ub-R48,
Lys
must be viewed as the important site for
polyubiquitination to render FBPase accessible to proteolysis.
Using
mutants defective in subunits of the 20 S proteasome we had shown that
this particle is involved in catabolite degradation of FBPase (12, 13) . In vitro studies had shown that
ubiquitinated proteins are degraded by the 26 S
proteasome(19, 20, 21) . To elucidate whether
the 26 S complex is needed for FBPase degradation, we assayed the
catabolite inactivation of the enzyme in strains carrying a
temperature-sensitive mutation in the CIM3 gene, which encodes
a regulatory subunit of the 26 S proteasome(47) . Cim3-1 mutant cells arrest the cell cycle at
nonpermissive temperature and accumulate Ub-Pro-gal, degradation
of which is ubiquitin-dependent. Pulse-chase analysis of FBPase during
glucose-induced degradation indicated that the enzyme is remarkably
stabilized in cim3-1 cells relative to the isogenic wild
type (Fig. 4). This documents that the entire 26 S proteasome
complex is necessary for catabolite inactivation to occur.
Figure 4: Glucose-induced degradation of FBPase depends on an intact 26 S proteasome. Pulse-chase analysis of FBPase degradation in CIM3 (A) and cim3-1 (B) mutant cells was done as described in the legend to Fig. 1.
The finding that catabolite inactivation of FBPase is triggered by ubiquitination pointed to the action of E2 (Ubc) enzymes involved in this process. Therefore we measured the degradation rate of FBPase after the addition of glucose in yeast strains deleted in various UBC genes and thus devoid of the respective activitiy of the Ubc enzyme. Pulse-chase analysis indicated that catabolite degradation of FBPase was unaffected in cells lacking a functional Ubc2 (Rad6), Ubc6, Ubc7, and Ubc10 (Pas2) protein (not shown). In contrast, the degradation was strongly inhibited in ubc1 and ubc4 ubc5 mutant strains (Fig. 5A). The half-life of FBPase was increased 2-fold in ubc1 cells and 4-fold in ubc4 ubc5 double mutant cells (Fig. 5B). Single deletions of either UBC4 or UBC5 led only to a weak but reproducible increase of the half-life of FBPase during the catabolite inactivation process (25 min and 20 min, respectively, versus 17 min in wild-type cells, not shown). This behavior might have been expected, because it is conceivable that the nearly identical Ubc4 and Ubc5 enzymes are functionally redundant(28, 29) .
Figure 5: Glucose-induced degradation of FBPase depends on UBC1, UBC4, and UBC5. A, pulse-chase analysis of FBPase degradation in wild-type (wt), ubc1, ubc4, ubc5, and ubc4 ubc5 mutants was done as described in the legend to Fig. 1. B, quantitation of an independent set of pulse-chase experiments analyzing FBPase degradation in wild-type, ubc1, and ubc4 ubc5 mutants.
When measuring the degradation of FBPase by assaying enzymatic activity, similar results were obtained (not shown). It should be noted that all ubc mutants showed the characteristic rapid loss of FBPase activity of about 50% during the first 15 min of catabolite inactivation. This is due to phosphorylation of FBPase (4, 5) and indicates that the transduction of the glucose-induced signal is not impaired in the ubc mutant cells. On the basis of genetic studies, it had been shown that Ubc1, Ubc4, and Ubc5 constitute a subfamily of ubiquitin-conjugating enzymes with overlapping functions (28) . This feature is nicely reflected in the catabolite degradation process of FBPase.
Based on the above described findings we assume the following model for catabolite inactivation of FBPase. The addition of glucose to yeast cells grown on a nonfermentable carbon source causes phophorylation (4, 5) and ubiquitination of the enzyme. The formation of a multiubiquitin chain targets FBPase for degradation through the 26 S proteasome, thereby changing the half-life of the enzyme from over 90 h on ethanol (3) to under 20 min (Fig. 5B). The ubiquitin-conjugating enzymes Ubc1, Ubc4, and Ubc5 and the glucose-inducible, newly synthesized protein(s) are necessary for FBPase inactivation to occur. It seems unlikely that the glucose-induced synthesis of a new protein concerns the provision of ubiquitin-conjugating enzymes, because activities of Ubc1, Ubc4, or Ubc5 are already present under the conditions of catabolite inactivation. Ubc1 is expressed in exponentially growing cells and with enhanced levels in stationary phase cells; Ubc4 is also expressed in exponentially growing cells, and Ubc5 is present in stationary phase cells(28, 29) .
The catabolite inactivation process of FBPase represents an ideal model to follow the fate of a protein from the signaling event to ubiquitin proteasome-mediated degradation. Respective studies are on the way.