From the Proteasomes are highly complex proteases
responsible for selective protein degradation in the eukaryotic cell.
26 S proteasomes consist of two regulatory 19 S cap complexes and the
20 S proteasome, which acts as the proteolytic core module. We isolated
six mutants of the yeast Saccharomyces cerevisiae
containing mutations in the 20 S proteasome Proteasomes are large proteinase complexes operating in the
cytoplasm and the nucleus of the eukaryotic cell. Two complexes are
found: (i) the cylindrically shaped 20 S proteasome (molecular mass
~700 kDa) and (ii) the larger 26 S proteasome (molecular mass ~1700
kDa), which is composed of the 20 S proteasome as the proteolytic core
and two additional 19 S cap complexes attached at both ends of the 20 S
cylinder (1). Proteasomes are the major tool for selective protein
degradation in the cytoplasm and nucleus of the eukaryotic cell
(2-4).
26 S proteasomes degrade ubiquitinated proteins in an
ATP-dependent reaction, whereas 20 S proteasomes are not
able to do so. It is thought that the 19 S cap complexes of the 26 S
proteasome, which consist of ATPases associated with various cellular
activities and non-ATPase subunits are responsible for recognition,
unfolding and transport of a substrate protein to the proteolytically
active 20 S core. Using artificial peptide substrates, the 20 S
proteasome exhibits at least three different proteolytic activities
characterized by the type of amino acid where cleaving occurs: the
chymotrypsin-like activity, the peptidylglutamyl peptide-hydrolyzing
(PGPH)1 activity and the
trypsin-like activity clipping at the C terminus of hydrophobic,
acidic, and basic amino acids, respectively (5-8).
The cylindrically shaped 20 S proteasome consists of a stack of four
rings, each containing seven subunits (4). In eukaryotes the outer
rings are built up of seven different Proteasome-mediated degradation is linked to many different pathways of
the cell. Proteasomes are implicated in stress response. They remove
abnormal proteins generated by heat stress or exposure of cells to
amino acid analogues (6, 14-16). Recent work demonstrated that
proteasomes even degrade abnormal lumenal proteins of the endoplasmic
reticulum, which have been transported to the cytoplasm prior to
degradation (17, 18). Proteasome-dependent degradation of
one or several defined substrate proteins has been found to constitute
a significant step in various regulatory pathways of the cell.
Proteasomes are involved in adaptation of metabolism by degrading
enzymes as fructose-1,6-bisposphatase (19-21) or ornithine decarboxylase (22) as well as transcriptional regulators like Gcn4,
which controls expression of metabolic enzymes (23). Proteasomes are
also linked to cell differentiation as had been shown for the
proteolytically unstable yeast MAT Most importantly, proteasomes play vital functions in the cell division
cycle (for reviews, see Refs.26-28). In yeast, the central cdc2/Cdc28
kinase is cell cycle phase specifically activated by association with
certain cyclin proteins. Such cyclin-dependent kinase
complexes (CDKs) are inactivated by proteasome-mediated degradation of
the respective cyclin proteins by this terminating distinct phases of
the cell cycle (29, 30). Two different pathways act in recruiting
cyclins for proteasomal destruction. G1 cyclins are tagged via Cdc34
complexed with Skp1, Cdc53, and the F-box protein Grr1 (SCF pathway)
(for review, see Ref. 31). In contrast, the anaphase-promoting complex
mediates B-type cyclin destruction in late mitosis (for review, see
Refs. 28 and 31). Proteasomal destruction is also needed for removal of
CDK inhibitor proteins as, e.g. the Sic1 protein (32). In
contrast to cyclin destruction here proteolytic degradation results in
activation of the respective CDK complex. Moreover, proteasomes degrade
other cell cycle controlling proteins, which are not associated with cdc2/CDC28 kinase; anaphase-promoting complex mediated degradation of
Pds1 (33), or Cut2 in Schizosaccharomyces pombe (34) and Ase1 (35) define essential transitions in mitosis.
In agreement with the fact that the Here, we present analysis of six pre3 mutant alleles; three
mutations (pre3-2, pre3-3, pre3-5) residing in the
substrate binding cleft of the Pre3 subunit exclusively caused defects
in the PGPH activity of the complex. Interestingly, these mutations led
to no detectable deficiency in proteasomal protein degradation.
In addition, no other significant phenotype was found for these class of mutants. Three other mutations residing at distinct positions of the
Pre3 subunit led to reduction of PGPH activity but also impaired other
peptide cleaving activities. Two of these mutations, pre3-1
and pre3-6, led to strong defects in proteasomal
proteolysis. These mutations in addition caused other significant
phenotypes; mutants exhibited slow growth, sensitivity to elevated
temperatures, impaired growth due to ectopical expression of cyclins,
and other cell cycle-related effects.
Materials--
DNA restriction and modifying enzymes and the
HindIII linker were obtained from Boehringer (Mannheim,
Germany). Peptide substrates were from Bachem (Basel, Switzerland).
AutoReadTM sequencing- and FluoroPrime labeling kit were
from Amersham Pharmacia Biotech (Freiburg, Germany). 5-FOA was from
Toronto Research Chem Inc. (North York, Canada).
Media--
Cells were grown either on YPD (1% yeast extract,
2% peptone, 2% glucose), or on minimal medium (0.67% Difco yeast
nitrogen base without amino acids) containing 2% glucose or 2%
ethanol as a carbon source and supplements (20 µg/ml uracil, 20 µg/ml histidine, 30 µg/ml leucine). Synthetic complete medium (CM)
was prepared as outlined in Ref. 39. Selection for uracil auxotrophic cells was performed on mineral medium containing 50 µg/ml uracil and
0.1% w/v 5-fluoroorotic acid (5FOA). Sporulation medium contained 1%
potassium acetate, 0.1% yeast extract, and 0.05% glucose.
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).
Manipulation of Microorganisms--
Growth, mating, sporulation,
and tetrad dissection of yeast cells was performed using standard
protocols (40, 41). Transformation of yeast cells with plasmids was
done by the lithium acetate method (42). Temperature sensitivity was
assayed by streaking cells on YPD agar plates at 37 °C and 30 °C
for control. Growth and manipulation of Escherichia coli
strains JM109 and DH5 Isolation of Mutants--
To isolate mutants containing
mutations in the PRE3 gene of the yeast 20 S proteasome
strain FABYSD17c was mutagenized with ethylmethansulfonate (36) and
thereafter screened for mutants defective in PGPH activity or
trypsin-like activity. PGPH activity was measured using overlay assays
with Cbz-Leu-Leu-Glu- Institut für Biochemie,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-type subunit
Pre3. Three mutations (pre3-2, pre3-3, and
pre3-5) which reside at the active site cleft of the Pre3
subunit solely caused reduction of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in
protein degradation nor to any other phenotype. However, the pre3-2 mutation strengthened phenotypes induced by other
20 S proteasomal mutations, indicating that the peptidylglutamyl
peptide-hydrolyzing activity has to fulfill some rescue functions. The
other three mutations (pre3-1, pre3-4, and
pre3-6) are located at diverse sites of the Pre3 protein
and caused multiple defects in proteasomal peptide cleaving activities.
pre3-1 and pre3-6 mutants exhibited significant defects in proteasomal protein degradation; they
accumulated ubiquitinated proteins and stabilized defined substrate
proteins as, e.g. fructose-1,6-bisphosphatase. In addition,
pre3-1 and pre3-6 mutant cells exhibited
pleiotropic phenotypes as temperature sensitivity and cell
cycle-related effects.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-subunits, and the inner rings
are composed of seven different
-subunits, each. X-ray structure
analysis of the ancestor 20 S proteasome from the prokaryote
Thermoplasma acidophilum, which is composed of only one type
of
- and
-type subunit revealed the particle to be a hollow
cylinder (9). The channel spanning the center of the complex is
subdivided in three cavities. Both outer cavities are formed by the
interfaces of the
and
rings, whereas the central cavity, which
contains the proteolytically active sites, is build up only from
rings (9). X-ray structure as well as mutational analysis uncovered the
N-terminal threonine of the
-type subunit to be essential for the
proteolytic activity of the Thermoplasma proteasome (9, 10).
Access to the active compartment is controlled by four narrow gates,
and proteins have to be unfolded before degradation. This general
design of the 20 S proteasome complex has recently been confirmed for
eukaryotes by x-ray structure analysis of the yeast 20 S proteasome at
a 2.4-Å resolution (11). This study in addition resolved the subunit arrangement of the eukaryotic 20 S proteasome, as well as structural details of individual subunits of the eukaryotic proteasome. X-ray structure in combination with lactacystin binding analysis determined the active sites of the yeast proteasome to locate at the
-type subunits Pre2, Pre3, and Pup1 (11). Utilizing site directed mutagenesis
of the essential N-terminal threonine residues of these subunits, the
individual peptide cleaving activities could be assigned to one of
these active subunits, each (12, 13); Pre2 confers the
chymotrypsin-like activity, Pre3 the PGPH activity, and Pup1 the
trypsin-like activity.
2 transcriptional repressor protein (24, 25).
-type subunits of the 20 S
proteasome contain the proteolytically active sites previous screens
for yeast cells with defects in 20 S proteasomal activities yielded
only mutants harboring mutations in subunits of this type: Mutations
residing in subunits Pre1 and Pre2 caused defects in chymotrypsin like
activity (6, 14), whereas mutants harboring mutations in the
proteasomal subunits Pre3 and Pre4 showed defective PGPH activity (15,
36). Interestingly mutants with impaired chymotrypsin like activity
(pre1 and pre2) exhibited strong reduction in
degradation of proteasomal substrates (6, 20, 24, 37, 38) whereas
pre4-1 mutants which lack the PGPH activity did not show
any decrease in protein degrading activity ((15) W. Hilt unpublished
data).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
was carried out following standard protocols
(43). Growth medium for E. coli strains was LB medium (43)
containing 50 mg/liter ampicillin if needed.
NA as substrate as described in Ref. 15.
Detection of the trypsin-like activity was performed with yeast
colonies which were grown at 30 °C on YPD or selective medium to a
minimum size of 3 mm and then replica-plated onto fresh agar plates
(YPD or selective medium) covered with sterile filter dishes. The
replica plates were incubated for 24 h at 30 °C. To
permeabilize cells, filters were transferred to second filters which
had been soaked with 1% toluol, 4% ethanol, 0.5 mM EDTA,
0.5 mM EGTA, 20 mM Tris/HCl, pH 9.0. After
drying, filters were fixed to Petri dishes covered with 1% agar and
overlaid with 10 ml of reagent mixture (1% agar, 45 °C, 20 mM Tris/HCl, pH 9.3, 100 µl of 250 mM
Cbz-Ala-Arg-Arg-
-naphthylamide) and incubated for at least 4 h
at 48 °C.
-Naphthylamide released from the peptide was detected
by its UV fluorescence or by formation of a red dye by coupling with
Fast Garnet GBC (10 ml of coupling mixture containing 0.2 M
citrate, 4% Tween, pH 4.4, and 100 µl of Fast Garnet GBC (50 mg/ml
in dimethyl sulfoxide)).
Preparation of Extracts, Enzyme Tests, and Immunological
Detection of Ubiquitin-Protein Conjugates--
For testing proteasomal
proteolytic activities, cells were grown in liquid YPD medium (1%
yeast extract, 2% peptone, 2% glucose) into early stationary phase.
Cell extracts were prepared by vortex mixing of 30% (v/v) cell
suspensions in 50 mM Tris/HCl, pH 7.5, with equal volumes
of washed glass beads for 3 min. The chymotrypsin-like activity and
PGPH activity were measured in the supernatants using Cbz-Gly-Gly-Leu-4-nitroanilide or Cbz-Leu-Leu-Glu-NA as substrates as described (6, 15). The trypsin-like activity was measured in 100 mM Tris/HCl, pH 9.3, at 48 °C using
Cbz-Ala-Arg-Arg-
NA as substrate. For immunological detection of
ubiquitin-protein conjugates, strains were grown in YPD medium into
early stationary phase and divided in two aliquots, which were further
incubated for 3 h at 37 °C or 30 °C. Preparation of extracts
and immunoblotting using an ubiquitin-protein conjugate antibody was
performed as described (15).
Pulse-Chase Analysis-- To investigate proteolytic degradation of fructose-1,6-bisphosphatase, immunoprecipitation and pulse-chase analysis were performed with the isogenic pre3 mutant strains YRG11, YRG12, YRG16, and the corresponding wild type strain WCG4a following methods described previously in Ref. 21. Western blot-based pulse-chase analysis was performed as followed: Cells were grown in glucose containing medium (YPD, CM) to A600 = 4-6. To induce fructose-1,6-bisphosphatase (FBPase) synthesis, cells were then transferred to glucose free ethanol medium (resulting A600 = 3) and incubated at 30 °C for 5 h. Thereafter, catabolite inactivation was induced by addition of glucose. Cells harvested at different chase times were broken, extracts were subjected to SDS gel electrophoresis, and FBPase contents were determined by immunoblotting.
Cloning of the pre3 Mutant Alleles-- Standard molecular biological methods were used (40). The pre3 mutant alleles were cloned by the gap repair method (44) or utilizing PCR amplification. For cloning by gap repair, the PRE3 3' flanking region (250-bp XmnI/NsiI fragment) and the 5' flanking region (248-bp NruI/EcoRI fragment) were joined by a EcoRI linker and cloned into the shuttle vector pRS316 (CEN6 URA3) (45). The resulting plasmid was linearized with EcoRI, and pre3 mutant strains were transformed. Uracil auxotrophic strains obtained were checked for the presence of repaired plasmids by plasmid rescue and restriction analysis. This approach was only successful in the case of strain C19 (pre3-3) allele. The other pre3 mutant alleles were therefore cloned by PCR amplification. The 1.4-kilobase pair NruI/SnaBI fragments containing the pre3 mutant loci were amplified using chromosomal DNA from strains C6, C13, FD80, and FD207 as templates and 5'-AAGGATCCCATACTTACCCTGCTCGCGA and 5'-AAAGGATCCGATACGTAGATACAGTCACCA as primers. Both primers contained a BamHI site at their 5' end, which was used for subcloning of the PCR fragments to the shuttle vector pRS315 (CEN6 LEU2) (45) yielding plasmids pRG21-pRG26.
Construction of Strains-- Standard yeast genetic and microbiological techniques (41) were used. Strains C6, C13, C19, FD80, and FD297 were generated by ethylmethansulfonate mutagenesis of strain FABYSD17C. A diploid pre3 knock-out mutant was generated as followed: A pRS316 based plasmid harboring a 1.4-kilobase NruI/SnaBI PRE3 fragment was gapped by HpaI digestion removing the complete PRE3 coding region and 6 bp from the 3'region as well as 47 bp from the 5'region. A 1.1-kilobase pair HindIII URA3 fragment excised from yEP24 was inserted using HindIII linker ligation. The deletion construct was excised as a BamHI/XhoI fragment. The diploid strain WCG4/4 was transformed with the linear fragment and uracil prototrophic strains were selected. To check correct deletion of PRE3 in strain YRG8, cells were sporulated and segregation of auxotrophic markers was determined. Spores derived from YRG8 tetrads showed a 2:0 segregation pattern (2 viable (ura3 PRE3); 2 inviable (ura3 pre3::URA3)). Correct integration was further confirmed by PCR. pre3 single mutant strains (YRG11-YRG16) isogenic with WCG4a were generated by a one-step gene replacement and checked for correct recombination by PCR. Strain YHI54/3211 is a spore clone derived from a cross of YRG12 against YHI29/1 (Table I).
Sequencing of Mutant Alleles-- The pre3 mutant alleles were sequenced by the dideoxy chain termination method using pRG21-pRG26 plasmids as templates. A double strand sequencing strategy was used as outlined in Ref. 36. Each allele was sequenced from the NruI site located in the PRE3 5' region up to 70 bp downstream from the PRE3 structural gene. Sequencing was performed on an A.L.F. automatic DNA-sequencer (Amersham Pharmacia Biotech) using the Auto-ReadTM sequencing kit and the FluoroPrimeTM labeling kit.
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RESULTS |
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Isolation of pre3 Mutants--
A series of mutants containing
mutations in the 20 S proteasome -type subunit Pre3 have been
isolated. Three pre3 mutants (pre3-1 to
pre3-3) had been found by screening
ethylmethansulfonate-mutagenized yeast cells for colonies with reduced
PGPH activity of the 20 S proteasome (36). An independent mutant screen
searching for cells that were defective for the trypsin-like and/or the
PGPH activity of the proteasome yielded two additional mutants allelic with PRE3, which were called pre3-4 and
pre3-5. McCusker and Haber (46) had performed a screen for
yeast mutants that showed resistance to a minimal concentration of
cycloheximide and in addition exhibited temperature sensitivity. One of
these mutants, crl21, uncovered to be allelic with
PRE3 (16), which in this work is referred to as
pre3-6.
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Isolation of pre3 Mutant Alleles-- The pre3-3 allele was rescued by the gap repair method. pre3-3 mutant cells were transformed with a linear DNA fragment containing 3'and 5' homologous DNA sequences of the PRE3 promotor and terminator region separated by the backbone of the yeast shuttle vector pRS316 (URA3 CEN6). Plasmids repaired by homologous recombination were isolated from plasmid-dependent uracil prototrophic cells. Interestingly, in an attempt to rescue the other pre3 alleles, the gap repair method failed. We suggest this result to be due to reduced rates of homologous recombination found at regions adjacent to centromeres (49) as is the PRE3 locus. Therefore, the other pre3 alleles were isolated by PCR using primers which matched at 5' and 3' regions of the PRE3 locus yielding a 1.4-kilobase DNA fragment containing the sequence coding for the respective pre3 mutant allele, and flanking regions containing 341 base pairs of the PRE3 promotor and 327 base pairs of the terminator sequence. The fragments were subcloned to the yeast centromere vector pRS315 (LEU2 CEN6) yielding plasmids pRG21 through pRG26 and sequenced.
Generation of Isogenic pre3 Mutants--
The diploid yeast
strain (yRG8) which contained a heterozygous chromosomal
pre3::URA3 deletion was transformed
with plasmids pRG21-pRG26 containing the pre3 mutant
alleles. Cells were sporulated and tetrads dissected. Colonies derived
from the spores were tested for uracil and leucine prototrophy by this,
yielding haploid strains which contained the lethal
pre3
::URA3 deletion, but were viable due to the presence of the respective pre3 pRG plasmid.
Proteasomal peptide cleaving activities of these
plasmid-dependent pre3 mutants were tested and
compared with proteolytic properties of the original pre3
mutant strains obtained by mutagenesis. No or only very slight deviations in the peptide cleaving activities were found (data not
shown).
Sequences of the Different pre3 Alleles--
Sequences of the
pre3 mutant alleles were determined (Fig.
2). pre3-1 contained one
single amino exchange (G15D) located at the N-terminal region of the S2
-sheet. The pre3-1 mutation locates in close proximity
to the active site cleft of the Pre3 subunit but also at a region
responsible for Pre3-Pup1
-
-contact. Three mutant alleles,
pre3-2, pre3-3, and pre3-5,
contained an exchange of glycine 47 (pre3-2 and
pre3-5: G47D; pre3-3: G47S). pre3-5 in addition contained a mutation (V59A) located centrally to the H1
helix. Glycine 47 is entirely conserved in all
-type subunits of the
20 S proteasome. It is located at a sharp bend between the C terminus
of
-sheet S4 and the N terminus of the H1 helix (11). This region
and especially the adjacent residues Arg-45 and Ala-49 are involved in
formation of the Pre3 substrate binding pocket (S1 pocket) (11). The
pre3-4 mutant allele harbored a single amino acid exchange
(G171D) residing centrally to the H4-S9 loop. This region is involved
in Pre3-Pre3 as well as Pre3-Pre4
-ring-
-ring interaction.
Interestingly, for the pre3-6 mutant sequence a short
deletion of 4 amino acids, Lys-85 through Glu-88, comprising the C
terminus of the H2 helix was found. This region is in close proximity
to the H2-S5 loop, which is part of the inner gates (the so-called
-annulus) of the yeast 20 S proteasome (11).
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pre3-1 and pre3-6 Are Deficient in Protein Degradation-- The series of pre3 mutants was tested for accumulation of ubiquitinated protein. This effect had been found as a typical phenotype of mutants defective in ubiquitin-proteasome-dependent protein degradation (6, 15, 16). Accumulation of ubiquitinated proteins significantly increased under certain stress conditions as heat stress or exposure of cells to amino acid analogues. Within the series of pre3 mutants, two strains YRG11 (pre3-1) and YRG16 (pre3-6) were found to accumulate ubiquitinated proteins at 30 °C and to stronger extent at 37 °C (Fig. 3A). In contrast as compared with wild type cells pre3-2 mutants (Fig. 3A) as well as pre3-3, pre3-4 and pre3-5 strains (data not shown) did not accumulate increased amounts of ubiquitinated proteins at both conditions.
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pre3-1 and pre3-6 Mutants Exhibit Growth Defects and Cell Cycle-related Defects-- Overexpression of certain cyclins acting in different phases of the cell cycle is not tolerated in pre3-1 and pre3-6 mutant cells. Ectopical expression of Gal promotor controlled plasmid encoded G1 cyclins CLN2 (Fig. 4A), CLN1 and CLN3 (data not shown) in strains YRG11 (pre3-1) and YRG16 (pre3-6) nearly completely abolished cell growth. Reduced growth was also found when overexpressing the S/G2 cyclin CLB5 (Fig. 4B) and to a minor extent the mitotic cyclin CLB2 in pre3-6 mutant cells (Fig. 4C). In pre3-1 mutant cells, CLB5 overexpression only slightly impaired cell growth (Fig. 4B), whereas no significant growth defect was observed when overexpressing CLB2 in pre3-1 cells (YRG11) (Fig. 4C). No such sensitivity to ectopically expressed cyclins were found in the other pre3 mutant strains: when overexpressing any cyclin in pre3-2, pre3-3, pre3-4, and pre3-5 strains, no additional reduction in cell growth as compared with wild type cells occurred (Fig. 4 and data not shown).
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DISCUSSION |
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We have isolated mutants carrying mutations in the 20 S proteasome subunit Pre3. Concerning their localization in the Pre3 structure as well as their influence on peptide cleaving activities of the 20 S proteasome, the isolated pre3 mutations can be subdivided in two groups. The first group of mutations (pre3-2, pre3-3, and pre3-5) residing near the active site cleft of the Pre3 subunit solely cause reduction of the PGPH activity of the 20 S proteasome. The second group of mutations (pre3-1, pre3-4, and pre3-6) locate at diverse sites of the Pre3 structure and lead to multiple defects in several peptide cleaving activities of the proteasome complex. Two of these mutations, pre3-1 and pre3-6, result in strongly impaired protein degradation via the proteasome pathway and cause other pleiotropic phenotypes. In contrast, pre3-4 mutants, which are only slightly reduced in trypsin-like and PGPH activity as well as all class I mutants, do not show any deficiency in proteasomal protein degradation or any other cellular phenotype.
X-ray structure and mutational analysis uncovered the Pre3 protein to
carry an active site, which is thought to be responsible for the PGPH
activity of the 20 S proteasome (11-13). This finding is confirmed by
the fact that pre3-2, pre3-3, and
pre3-5 mutations lead to sole reduction of the PGPH
activity of the proteasome. All three mutants contain a mutation of
glycine 47. This residue is completely conserved in all -type
subunits analyzed so far and is part of the highly conserved so called
GD-box spanning from Gly-47 to Asp-51. The strong conservation
indicates that this sequence motif constitutes a structure essential
for the function of the proteasome. X-ray structure data suggested that Gly-47 may essentially contribute to substrate cleavage. A fully occupied solvent molecule, which is thought to serve as the nucleophile attacking substrate molecules during hydrolysis, has been found in
close vicinity to Gly-47. In addition, x-ray structure analysis of
T. acidophilum (9) and S. cerevisiae (11) 20 S
proteasomes treated with the inhibitor acetyl-Leu-Leu-norleucinal
detected a hydrogen bond between Gly-47 and the inhibitor molecule.
Moreover, Gly-47 locates to the center of a small loop between the S4
-sheet and the H1
-helix (11). The adjacent residues Arg-45 and
Ala-49 form the bottom of the binding cleft of the Pre3 subunit.
Moreover, residue Arg-45 is thought to comprise the site responsible
for specific binding of substrates containing an acidic amino acid at
the cleavage site (11). Exchange of Gly-47 against larger amino acids
may disturb coupling of the nucleophilic water molecule to the binding
cleft. In addition, such new residue may reach into the binding cleft
and sterically hinder substrate binding. One may alternatively imagine
that exchange of Gly-47 against other residues may result in distortion
of the sharp bend formed by Gly-47. By this, disarrangement of Arg-45
and Ala-49 may occur, which finally may lead to a strong deficiency in
substrate binding. pre3-5 mutants in addition contained an
exchange of valine 59 against alanine. However, the pre3-5
mutant showed the same phenotype as pre3-2 cells, which
carry the identical exchange, G47D, indicating that the additional
mutation of pre3-5 does not cause any effect on 20 S
proteasome function.
Data clearly indicate that the pre3-2, pre3-3,
and pre3-5 mutations lead to a specific effect which is
limited to the Pre3 subunit of the 20 S proteasome. Interestingly,
though almost completely lacking the proteasomal PGPH activity, no
detectable defect on protein degradation nor any other additional
cellular phenotype was found in these mutants. Identical results (loss
of PGPH activity but no detectable defect in proteasomal protein
degradation) had previously been obtained for pre3T1A
mutants containing an exchange of the essential N-terminal threonine
(13) as well as pre4-1 mutants lacking 15 amino acids at
the C terminus of the Pre3 -ring neighbor Pre4 (15). It had been
discussed that mutants, although completely deficient in cleaving the
artificial peptide substrate Cbz-Leu-Leu-Glu-
NA, may exhibit some
residual activity for hydrolysis of peptide bonds within natural
substrate proteins (15). This leakage model may now be excluded due to
the fact that three structurally quite different mutations such as
pre4-1, pre3T1A, and pre3G47D(S) lead
to the same phenotypic effect. The data strongly indicate that loss of
the PGPH activity does indeed not result in any deficiency in protein
degradation via the proteasome. In case proteasomes lacking the PGPH
activity were indeed fully active, the question arises why this
activity has not been lost during evolution. Interestingly, pre4-1 (15) or pre3T1A (13) when combined with
pre1-1 or pre2 mutations, which lead to
defective chymotrypsin-like activity, significantly enhanced
pre1-1- or pre2-induced defects. In agreement with these results, we now found that the pre3-2 mutation
considerably strengthened pre1-1-induced temperature
sensitivity. These data indicate that, when lacking one of the other
activities, the PGPH activity may become important. Therefore, the
necessity for the PGPH activity may rest in a redundancy function.
Alternatively, it cannot be excluded that conditions may exist which
require a proteasome with all proteolytic sites being active.
Similarly to pre3-2, pre3-3, and
pre3-5 mutations, the pre3-4 mutation did not
cause any defect in protein degradation nor any other phenotype. The
pre3-4 mutation in addition to a defect in the PGPH
activity caused reduction of the trypsin-like activity. However, both
activities are reduced to only minor extents, and this may be the
reason for the fact that pre3-4 does not lead to defective
protein degradation. The mutation (G171D) resides in a region that is
in close vicinity to the Pre3 and Pre4 subunits of the neighbor
-ring. Even though pre3-4 more strongly influenced the
trypsin-like activity, the mutation is not in close contact to the Pre3
-ring neighbor subunit Pup1 (11), which harbors the active site
responsible for trypsin-like activity (13). As pre3-4
mutants showed normal chymotrypsin-like activity and in addition
exhibited no other phenotype typically found in cells defective in
proteasome function, this mutation does not seem to impair general
formation of the 20 S proteasome complex. We assume that
pre3-4 either influences the Pup1 subunit by a long distance effect or disturbs the local arrangement of the Pre3 Pre4 Pup1
subunit cluster
The pre3-1 mutation (G15D) locates to a region that is in
close contact to the Pre3 active site cleft as well as the domain responsible for interaction of the Pre3 protein with its -ring neighbor Pup1. Therefore, reduction of both activities, the PGPH and
the trypsin-like activity found in pre3-1 mutants, can most probably be explained by a pre3-1-induced structural
alteration influencing the two active sites located within these
subunits.
pre3-6 harbors a short deletion (Lys-85 to Glu-88) of the
C-terminal part of the H2 helix. This region is directly connected to
the H2-S5 loop, which forms the Pre3 part of the -ring gate (the so
called
-annulus; Ref. 11) located at the central channel of the 20 S
proteasome. pre3-6 causes significant reductions of all
three peptide cleaving activities. We therefore speculate that
pre3-6 may lead to a severe alteration of the complete 20 S
proteasomal
-ring structure or may impair assembly of the 20 S
complex.
Both mutations, pre3-1 and pre3-6, cause a deficiency in protein degradation as proven by stabilization of undefined ubiquitinated proteins as well as defined substrates of the proteasome pathway. In addition, these mutations resulted in pleiotropic phenotypes as temperature sensitivity and distinct cell cycle related effects. Although nearly identical, phenotypes seem to be slightly stronger expressed in pre3-6 mutants. We assume that both mutations lead to a general defect in proteasomal protein degradation. As indicated by the enhanced accumulation of ubiquitinated proteins at 37 °C, the temperature sensitivity of pre3-1 and pre3-6 cells can most probably be explained by insufficient removal of abnormal proteins generated under such heat stress conditions. In addition, in contrast to certain 19 S regulatory complex mutants (cim3/cim5) which, at restrictive temperature, stop growth in the G2/metaphase (54), temperature sensitivity of pre3-1 and pre3-6 cells was proven to be a cell cycle-independent event.
At permissive temperature, pre3-1 and pre3-6 mutants contained increased amounts of 2n DNA indicating a disturbed time course of cell cycle phases. This result may be explained by stabilization of certain cell cycle regulators. Cells expressing truncated proteolytically stabilized versions of G1 cyclins Cln3 or Cln2 (55, 56) or cells that due to a grr1 mutation show defective Cln degradation (57) prematurely enter S phase with a reduced G1 phase. In addition, progress through and exit from mitosis essentially requires proteasomal destruction of B-type cyclins (58, 59) and other mitotic regulators as Pds1 (33) or Ase1 (35). Therefore, enhanced amounts of 2n DNA containing cells detected in pre3-1 and pre3-6 mutant strains may be assigned to premature entry into a new cell cycle as well as delayed transition through mitosis. However, start into a new cell cycle also requires destruction of the CDK inhibitor Sic1 via the proteasome. Therefore, premature entry into S-phase may not occur in pre3-1 and pre3-6 cells. This view is supported by the fact that no significant decrease in cell size was observed for pre3-1 and pre3-6 mutants as is normally found for budding yeast cells showing accelerated transition through G1.
Ectopical expression of G1- and B-type cyclins in pre3-1 and pre3-6 mutant cells resulted in slightly to significantly reduced growth. In the case of B-type cyclin overexpression, the growth defect observed can most probably be attributed to impaired inactivation of B-type cyclin-dependent CDKs by this strengthening delayed exit from mitosis. Interestingly, stronger growth defects were found when overexpressing G1 cyclins in pre3-1 and pre3-6 mutant cells. However, no stringent necessity for G1 cyclin destruction could be detected so far (57). Therefore, sensitivity to ectopical expression found in pre3-1 and pre3-6 cells cannot be assigned to a defined process yet. Overexpression of Cln2 in wild type cells was found to result in hyperpolarization of cortical actin and secretion indicating that Cln1/2-dependent CDKs trigger polar bud growth (60). In agreement with these results, impaired degradation of Cln1/2 in grr1 mutant cells resulted in the formation of elongated cells (57). Therefore, appearance of misshapen cells with strongly elongated buds in cultures of pre3-1 and pre3-6 mutants may be explained by delayed degradation of Cln1/2 proteins.
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ACKNOWLEDGEMENTS |
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We thank C. Mann for friendly reception and support of R. G. during his stay in Gif-sur-Yvette. We also thank R. J. Deshaies, M. Kirschner, and W. Seufert for support with plasmids and M. Groll and R. Huber for providing structure data.
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FOOTNOTES |
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* This work was supported by a Program de Coopération Scientifique grant for scientific exchange from the Deutsche Akademische Austauschdienst, Bonn; by the Fonds der Chemischen Industrie, Frankfurt; and by the German Israeli Foundation for Scientific Research and Development, Jerusalem.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: Institut für Biochemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany. Tel.: 49-711-685-4388; Fax: 49-711-685-4392; E-mail: hilt{at}po.uni-stuttgart.de.
1
The abbreviations used are: PGPH,
peptidylglutamyl peptide-hydrolyzing; Cbz, carbobenzoxyl; 5FOA,
5-fluoroorotic acid; -gal,
-galactosidase; FBPase,
fructose-1,6-bisphosphatase; CDK, cyclin-dependent kinase
complex; bp, base pair(s); CM, complete medium; PCR, polymerase chain
reaction;
NA,
-naphthylamide.
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REFERENCES |
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