From the
The cloning, expression, and biochemical characterization of an
essential gene of Saccharomyces cerevisiae that encodes for a
new member of the TBP1-like subfamily of putative ATPases are
described. The protein is 72% identical at the amino acid level to
subunit four (S4) of the human 26 S protease and 73% identical to
Schizosaccharomyces pombe MTS2 gene product. The purified,
recombinant protein, designated Yhs4p, has an estimated molecular mass
of 49 kDa and exhibits a Mg
The 20 S proteasome is a widely occurring intracellular,
multicatalytic protease of 700 kDa (for review, see Ref. 1) composed of
24-28 subunits of 20-30 kDa encoded by the
``proteasome gene family'' (for review, see Ref. 2). Several
hetero-oligomeric protein complexes of about 10 subunits ranging from
40 to 110 kDa (
The discovery of the
human immunodeficiency virus Tat-binding protein TBP1
(12) unveiled a new subfamily of putative ATPases of
approximately 45-50 kDa containing a single ATP binding site
called ``P-loop'' domain
(13) . This
``TBP1-like'' subfamily initially included the transcription
activator Tat-binding protein TBP7
(14) , a modulator of human
immunodeficiency Tat-mediated transactivation MSS1
(15) and the
yeast protein SUG1
(16) . More recently, this subfamily has been
expanded to include components of the regulatory subunits of the 26 S
protease. Subunit four (S4) of the 26 S protease is a putative ATPase
that shares identity with TBP1, TBP7, SUG1, and MSS1
(17) .
Partial sequence analysis proved that MSS1 is subunit 7
(18) and TBP7 is subunit 6
(19) of the 26 S protease.
Therefore, the putative ATPases among the larger subunits of the 26 S
protease may have a dual role in the regulation of transcription and
proteolysis. Alternatively, they may only function as 26 S protease
subunits, and the regulation of transcription may be a consequence of
the selective proteolysis catalyzed by the complex
(19) .
Three other members of this TBP1-like subfamily MTS2 from
Schizosaccharomyces pombe (20) and CIM5 and CIM3 from
Saccharomyces cerevisiae (21) are involved in the
regulation of the yeast proteasome in vivo. MTS2 is 75%
identical to S4 of the human 26 S protease. CIM3 is identical to SUG1,
and CIM5 is 33% identical to S4. The yeast 20 S proteasome, known as
yscE proteinase, has been extensively characterized (for a review, see
Ref. 22). The yeast 26 S protease has not yet been purified. Therefore,
no information is available on its subunit composition.
We have
cloned and expressed an S. cerevisiae essential gene encoding
the 49-kDa protein Yhs4p that is 72% identical to S4 of the human 26 S
protease and is a member of the TBP1-like subfamily of putative
ATPases. We present the strategy of expression and purification of
Yhs4p that allowed a characterization of its ATPase activity. Yhs4p is
the first member of the TBP1-like subfamily on which an ATPase activity
is demonstrated in vitro. The amino terminus of Yhs4p is
involved in negative modulation of the ATPase activity.
The observation that the
amino-terminal truncated Yhs4p is a more active ATPase than the
complete molecule (Fig. 6) suggests that the amino-terminal
region of Yhs4p has a negative regulatory function. A distinctive
feature of the amino-terminal region is the presence of two clusters of
positively charged amino acid residues (See Fig. 1). To learn if
these clusters were involved in the observed negative modulation, we
studied the effect of several polyanions and two polycations on the
ATPase activity of complete and amino-terminal truncated Yhs4p. Both
Mg
We have cloned and expressed a yeast cDNA encoding a new
member of the TBP1-like subfamily of putative ATPases, designated
Yhs4p. Disruption of YHS4 gene (Fig. 3) shows that the
production of Yhs4p is essential for cell viability. In previous
studies, using strategies of gene disruption/complementation in yeast
(12, 14, 15) and co-transfection/over
expression in human cells
(16, 20, 21) , some
members of this subfamily have been identified as transcriptional
regulators
(12, 14, 15, 16) , while
others were proposed to be essential for the function of the 26 S
protease involved in ATP/ubiquitin-dependent proteolytic pathway
(20, 21) . Initially the TBP1-like subfamily of putative
ATPases was composed of proteins identified as transcriptional
regulators (TBP1, TBP7, MSS1, and SUG1). Recent findings contributed to
expand our understanding on the biological function of members of this
subfamily. (i) Subunit 4 of 26 S protease is a member of this subfamily
(17) , and (ii) MSS1 and TBP7 are identical to subunits of the
26 S protease
(18, 19) . These findings suggested that
alterations in the activity of the 26 S protease may alter the pattern
of transcriptional regulation
(19) . It is also possible that
some members of this subfamily may play a dual role in transcriptional
regulation and in proteolysis catalyzed by the 26 S protease. It is
essential to characterize their biochemical activities to gain further
insight into their biological role. No member of this subfamily has
been biochemically characterized yet. Their primary protein structure
contain four domains that are characteristically found in ATP/GTP
binding proteins
(13, 31) and in DNA/RNA helicases
(32, 33) . These domains are highly conserved and
cluster at a constant distance of approximately 250 residues from the
amino-terminal end. Yhs4p, MTS2, and S4 share the highest identity
(
Yhs4p contains four cysteine residues (Fig. 1). The
cysteine residue closest to the carboxyl terminus is conserved in all
members of TBP1-like subfamily (Fig. 2, central diagram). The ATPase activity of the regulatory subunit
of the 26 S protease PA-700 is inhibited by low concentrations of the
sulfydryl reagent N-ethylmaleimide
(8) . Similarly,
ATPase activities of complete and amino-terminal truncated Yhs4p were
90% inhibited by 50 µ
M N-ethylmaleimide (not
shown), suggesting that one or more of its four cysteine residues are
essential for the activity.
Yhs4p has substrate specificity
(Fig. 8) and kinetic parameters (Fig. 5) resembling those
described for the ATPase activity of the 26 S protease
(10) .
The high identity of Yhs4p with human S4 of the 26 S protease
(17) and S. pombe MTS2 involved in regulation of the
yeast proteasome in vivo (20) suggests that this
protein may have a role in the regulation of the proteasome in the
yeast S. cerevisiae. The similarity between the ATPase
activity of Yhs4p reported here and 26 S protease suggests that the
role may be as a catalytic subunit for the hydrolysis of ATP coupled to
the hydrolysis of ubiquitin conjugates by the 26 S protease. Other
putative activities that may be attributed to Yhs4p based on observed
sequence homology are the function of a protein kinase, DNA/RNA
helicase, and GTPase. These putative activities may require
oligomerization of Yhs4p or interaction with the appropriate regulatory
protein(s).
While this work was in progress, the S. cerevisiae gene designated YTA5 was cloned and sequenced
(40) and was
shown to encode a protein identical to Yhs4p. This work, however, shows
a direct biochemical activity for the protein product of the gene as an
ATPase and proves that the gene is essential. The production of Yhs4p
as a fusion protein enabled its purification and the partial
characterization of its ATPase activity. To reiterate, no other member
of the TBP1-like subfamily of putative ATPases has been characterized
yet in terms of its ATPase activity or any other putative activity
predicted by its primary sequence.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) L17040.
H. A. L. thanks Dr. Benjamin Kaminer for continued
support.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dependent ATPase activity
with nucleotide specificity and K
for ATP
similar to those exhibited by the human 26 S protease. The observed
ATPase activity was reduced by 73% upon the introduction of point
mutation K229Q in the ``P-loop'' domain of the ATP-binding
site relative to the nonmutated form of the protein. This is the first
direct biochemical evidence supporting the putative ATPase activity of
a member of the TBP1-like subfamily. Furthermore, the experimental
results demonstrate a regulatory function for the amino-terminal region
of the molecule. The amino-terminal truncated form of Yhs4p lacking two
clusters of positively charged amino acids exhibits a greater ATPase
activity. The ATPase activity of both the truncated and complete forms
of Yhs4p is stimulated by polyanions. Polylysine partially inhibits the
ATPase activity of the amino-terminal truncated form having no
observable effect on the complete protein. N-Ethylmaleimide
inhibits the ATPase activity of both forms of Yhs4p. We propose that
Yhs4p ATPase may play an essential role in the regulatory function of
the proteolytic activity of the yeast 26 S protease.
700 kDa) have been described as activators of the
20 S proteasome. Among the activator complexes are CF-1
(3) and
``ball''
(4) from rabbit reticulocyte, the µ
particle
(5) from Drosophila, and PA-700
(6) from bovine blood cells. These complexes are called
``19 S cap complex''
(7) and assemble with the 20 S
proteasome to generate the 26 S protease. The similarities and
differences among these complexes are still under investigation
(4, 8) . However, some functional aspects of their
interaction with the 20 S proteasome have been established (for a
review see Ref. 7). (i) The assembly between the 20 S proteasome and
each of the activator complexes is coupled to ATP hydrolysis, and only
CTP substitutes for ATP
(3) . (ii) The 26 S protease exhibits an
ATP-dependent proteolytic activity on ubiquitin-conjugated proteins,
which is not observed in the 20 S proteasome. All four nucleotides
stimulate proteolysis of ubiquitin conjugates by the assembled 26 S
protease
(9) , and are hydrolyzed by it
(10) . Therefore,
the subunits of the activator complexes confer ATP dependence and
ubiquitin recognition to the proteolytic core provided by the 20 S
proteasome subunits
(9, 11) .
Yeast Strains and Analysis of Mutants, Saccharomyces
cerevisiae
Diploid strain W303 ( leu2, his3, ade2, trp1,
ura3) was used for the disruption of the gene YHS4.
Wild-type and mutants were grown in 1% yeast extract, 2% peptone, 2%
glucose medium
(23) . Cells were transformed using the lithium
acetate method
(24) and then grown on minimal media
supplemented with the appropriate nutritional requirements
(25) . The YHS4 gene, deleted in the 5` end of the open
reading frame, was cloned by serendipity while screening a yeast cDNA
gt11 library. The cDNA was used as a probe for screening a yeast
genomic library in YEp13 kindly donated by Dr. Michael Douglas. A 2.3
kb
(
)
genomic fragment containing a complete open
reading frame was isolated. To interrupt the YHS4 gene, a
1.3-kb EcoRI fragment of the
gt11 clone was subcloned
into the EcoRI site of pBluescript. The resulting plasmid was
linearized with HpaI, which cuts after 0.45 kb from the 5` end
of the insert, and LEU2 gene was cloned into the site. The
construct was excised by HindIII leaving 0.43 and 0.75 kb at
the 5` and 3` ends, respectively. The above HindIII fragment
was used to interrupt the YHS4 gene in diploid W303 cells as
described previously
(23) . Colonies that grew on minimal medium
lacking leucine were analyzed by polymerase chain reaction, sporulated
on acetate medium, and subjected to tetrad dissection.
Mutation of the P-loop Motif and Subcloning
Two
primers were synthesized to mutate the lysine residue
(Lys) in the P-loop motif to either histidine or
glutamine. The sense primer was 5`-CCC GGT ACA GGT CAT ACA TTG
CTA GCA AAG-3`, and the antisense primer was 5`-TAG CAA TGT CTG ACC TGT ACC GGG TG-3`. The mutation was generated as described
previously
(26) by polymerase chain reaction overlap extension
using the 2.3-kb genomic fragment as template and the 5` primer ATA GAA
AAT GGA TCC GGA CAA GGT GTA TCA TCT G and the 3` primer GCA
TTA CTT GGA TCC GTA AAT CTA TTA TAG AAA AAT GTA. Each primer
carries a BamHI site ( underlined) inframe with the
corresponding BamHI site of the pGEX-2T plasmid. The mutant
used in the expression and ATPase activity assays presented in this
report is AAG to CAG in codon 229 of the open reading frame,
corresponding to K229Q in the protein sequence. The reading frame of
the wild-type YHS4 gene was adapted by polymerase chain
reaction amplification with the same 5` and 3` primers. Wild-type and
mutated YHS4 gene were subcloned into the BamHI site
of pGEX-2T following standard procedures
(27) . A 1.3-kb
EcoRI fragment of the
gt11 clone was subcloned inframe
into the EcoRI site of the pGEX-2T. Escherichia coli cells BL21(DE3)pLysS, transformed with the wild-type, mutated, and
truncated YHS4 genes were grown at 37 °C in 100 ml of
LB/ampicillin medium in a 500-ml flask with shaking at 300 rpm. At a
cell density of 0.4 OD units at 600 nm,
isopropyl-1-thio-
-
D-galactopyranoside (1 m
M final concentration) was added and growth was continued for 2 h at
30 °C. Cells were sedimented at 3,000
g at 4
°C for 15 min, resuspended in 5 ml of 137 m
M NaCl, 2.7
m
M KCl, 4.3 m
M Na
HPO
7H
O, 1.4 m
M KH
PO
, pH 7.3 (PBS), and disrupted by three
passages through a French press. The homogenates were then centrifuged
at 30,000
g for 20 min at 4 °C, pellets were
resuspended in 5 ml of PBS, and supernatants were absorbed for 5 min at
4 °C with 0.5 ml of GSH-agarose (50% (v/v) slurry in PBS).
GSH-agarose beads were collected by microcentrifugation for 5 s and
washed 3 times with 1 ml of PBS. Washed beads were resuspended for 5
min at 4 °C with 0.5 ml of 50 m
M Tris-HCl, pH 7.3,
containing 5 m
M GSH. Beads were spun down as above, and
supernatants containing the fusion proteins were concentrated 5-fold by
ultrafiltration in a Centricon-10 (Amicon) cartridge. ATPase activity
was assayed immediately after this step in a basic medium (50 µl)
containing 50 m
M Tris-HCl, pH 7.6, 5 m
M dithiothreitol, 5 m
M MgCl
, and 50 µ
M ATP/[
-
P]ATP (1,000-2,000
cpm/pmol) at 37 °C. Nucleoside triphosphate, EDTA, polycations
(polylysine and polyarginine), polyanions (M13 single-stranded DNA,
yeast total RNA, poly-Glu-Ala-Tyr (6:3:1), and pBluescript
double-stranded DNA), and N-ethylmaleimide were added to this
basic medium where indicated. The reaction was initiated by the
addition of 20 ng of fusion protein and ended by the addition of 500
µl of 10% trichloroacetic acid.
PO
released from [
-
P]ATP was determined
as described
(28) . Protein was determined by the method of
Lowry et al. (29) . SDS-PAGE gels
(30) were
stained with Coomassie Blue R-250.
Cloning and Characterization of Yhs4p
cDNA
Initial cloning efforts resulted in the isolation of a
yeast cDNA containing a 1.3-kb EcoRI fragment encoding a
amino-terminal truncated protein of 387 residues (43 kDa) with homology
to the TBP1-like subfamily. The cDNA truncated in the 5` region was
used to probe a yeast genomic library resulting in the isolation of a
2.33-kb fragment from a yeast genomic library that encodes a protein of
437 residues (49 kDa). The 1.3-kb cDNA fragment lacks the
5`-untranslated region and 150 base pairs of the open reading frame
encoding the first 50 amino acid residues from the amino-terminal. The
deduced protein sequence designated Yhs4p is shown in Fig. 1.
Underlined are the 50 amino acid residues that are absent in
the amino-terminal truncated molecule. Two clusters of positively
charged amino acids, KKKKK
and
RKKRK
, are present at the amino terminus. The
sequence also contains a P-loop domain
GAPGTGK
, found in ATP/GTP binding proteins
(13) , two additional domains,
DEIDAIG
and
MATNKIE
, characteristic of GTP
binding proteins
(31) , and the
LIRPGRIDR
domain homologous to the domain VI of a subfamily of
ATP-dependent DNA/RNA helicases
(32, 33) . These four
domains, characteristic of ATP/GTP-binding proteins and ATP-dependent
DNA/RNA helicases, and the cysteine residue (Cys
) near
the carboxyl terminus of the sequence are conserved in all members of
the TBP1-like subfamily. Fig. 2 depicts the sequence of these sites,
designated with the roman numerals from I to IV, and their position in
the primary structure of the members of this subfamily. Interestingly
these sites cluster at approximately 250 amino acid residues from the
amino termini and span 100 amino acid residues. Yhs4p sequence is 73%
identical to MTS2 and 72% identical to S4 of 26 S protease in the whole
span of their primary sequence comprising around 450 amino acid
residues. The identity is lower (40-50%) in the first 25 amino
acid residues at the amino-terminal region. These structural features
suggest that MTS2 and Yhs4p are yeast homolog to human S4. The identity
of Yhs4p with other members of the TBP1-like family is lower
(50-60%), and it is restricted to the 200-300 amino acid
residues containing domains I-IV, characteristic of several
NTP-binding proteins. The amino-terminal region of these proteins has
only a 6-7% identity with Yhs4p. The consensus sequences of
domains I-VI in the TBP1-like subfamily and the proposed function
of similar sites in members of other NTP binding families are
summarized in the lower part of Fig. 2.
Figure 1:
Predicted amino acid sequence encoded
by YHS4. The protein sequence of Yhs4p is presented using the
single letter amino acid code. Underlined in italics are the 50 amino acid residues that are missing in the
amino-terminal truncated molecule. The cysteine residues are
underlined in boldface letters. Shaded are
the two clusters of positively charged amino acid residues. Double
underlined in boldface letters are the domains present in
ATP/GTP-binding proteins and in ATP-dependent DNA/RNA helicases . The mutation K229Q is shown by the
arrow.
Figure 2:
Primary sequence identity and conserved
domains in members of the TBP1-like subfamily of ATPases. The sequences
of the four domains ( I-IV) conserved in members of the
TBP1-like subfamily of ATPases are shown in the upper portion
of the diagram. In the central portion the bars represent the
primary structure of the proteins. Empty boxes indicate the
localization of the four domains in the primary structure. Circle near the carboxyl termini indicates the position of the conserved
cysteine residue. Numbers on top of the bars indicate
percent of sequence identity of the region with the corresponding
region in Yhs4p. The lower part shows the consensus sequence for
domains I-IV in this subfamily, and the interactions proposed for
similar domains in other NTP-binding molecules. In brackets are all possible amino acids found in that particular position in
all members of the subfamily.
Gene Disruption and Tetrad Analysis
YHS4 gene was disrupted with the S. cerevisiae LEU2 gene.
Polymerase chain reaction analysis of colonies that were prototrophic
for leucine was used to confirm the presence of the yhs4::LEU2 disruption allele (data not shown). Eight Leuisolates were sporulated, and tetrads were dissected. In every
case but two, the four spores segregated in a ratio of two viable to
two nonviable (Fig. 3). The viable spores were auxotrophic for leucine
and did not carry the marker for the disruption.
Expression of Yhs4p
Fragments encoding the
complete Yhs4p, the amino-terminal truncated protein, and a protein
with a K229Q mutation in the P-loop domain were subcloned in the vector
pGEX-2T and expressed to render fusion proteins with glutathione
S-transferase. Wild-type and P-loop-mutated Yhs4p proteins are
well expressed in E. coli cells (Fig. 4, Whole cell after induction). However, most of
the expressed proteins remain in the pellet fraction obtained after
disruption and subsequent sedimentation (Fig. 4,
Pellet). Purification of the fusion proteins that remain
soluble after sedimentation (Fig. 4, Supernatant) was
undertaken using glutathione-agarose affinity chromatography. The
affinity-purified supernatants (Fig. 3, GSH- agarose purified supernatant) showed no protein in the
supernatant from control cells ( lane 1) and a single
77 kDa band in supernatants from cells expressing the wild-type
( lane 2) and P-loop-mutated ( lane 3) fusion proteins. 77 kDa is the expected molecular mass
for a fusion product between Yhs4p of 49 kDa and glutathione
S-transferase of 28 kDa. Amino-terminal truncated protein was
also expressed and purified as above (data not shown). As expected,
amino-terminal truncated Yhs4p travels slightly faster than the
wild-type and mutated proteins (Fig. 5). The yield of affinity-purified
fusion proteins varied between 0.5 and 3 µg/300 ml liquid culture.
Figure 4:
Expression of wild-type and K229Q mutated
Yhs4p proteins in E. coli. Complete and P-loop-mutated
YHS4 DNAs were subcloned in pGEX-2T plasmid inframe with the
glutathione S-transferase gene and expressed as described
under ``Experimental Procedures.'' E. coli cells
that were not transformed, called control cells,
( lane 1) cells that were transformed with the complete
( lane 2) and with the mutated ( lane 3) Yhs4p gene and fractions of these cells were resolved in 15% SDS-PAGE
gel. Whole cells (15 µg of protein) were analyzed before and after
induction with isopropyl-1-thio--
D-galactopyranoside.
Equal volumes of Pellet fraction containing 15 µg of
protein and Supernatant fraction containing 5 µg of
protein and 30 µl of the GSH- agarose purified supernatants (10 ng protein for lanes 2 and 3) from cells after induction with
isopropyl-1-thio-
-
D-galactopyranoside were loaded in the
gel. The arrow on the right indicates the positions of the
expressed fusion proteins.
Figure 3:
Tetrad analysis of yhs4::LEU2 disruption allele. The disruption allele was constructed and
spores were analyzed as described under ``Experimental
Procedures.'' All viable spores were phenotypically
Leuconfirming that the yhs4::LEU2 disruption allele is lethal.
Kinetic Parameters and Effectors of Yhs4p ATPase
Activity
The affinity-purified Yhs4p is a
Mg-ATPase with a K
for
ATP of 5 µ
M and a V
of 7 pmol of
ATP/min/µg of protein (Fig. 6). The amino-terminal truncated
protein exhibited the same K
value but a
V
of 20 pmol of ATP/min/µg of protein.
Mutational analysis of the P-loop domain G XXXXGKT (where X can be any amino acid) of several ATP/GTP-binding proteins
revealed that the lysine residue is essential for optimal activity. It
was recently reported that the K155Q mutation in the P-loop domain of
the
subunit of E. coli F
-ATPase impairs its
catalytic activities
(34) . The mutation K229Q in P-loop of
Yhs4p inhibited 73% of the wild-type ATPase activity, suggesting that
the lysine residue is required for optimal function of the ATPase. No
ATPase activity was observed in GSH-agarose purified supernatants from
control cells. The ATPase activity of complete and N-truncated
Yhs4p was 95% inhibited by EDTA and by 50 µ
M N-ethylmaleimide (not shown).
-ATPase activities were stimulated by polyanions.
Single-stranded DNA, double-stranded DNA, RNA, and poly(Glu-Ala-Tyr)
produced a 4-6-fold stimulation of the ATPase activity in the
complete protein, and a 1.5-2-fold stimulation in the
amino-terminal truncated protein (Fig. 7). The polyanions
produced maximal effect at a concentration of 10 µg/ml under the
experimental conditions used in the ATPase activity assay. The
polycation polylysine had only a small inhibitory effect on the ATPase
activity of the complete Yhs4p and inhibited 48% the activity of the
amino-terminal truncated molecule (Fig. 7). Polyarginine elicited
a similar pattern of inhibition on both forms of Yhs4p (not shown).
These data suggest that the positively charged clusters at the amino
terminus of the protein are involved in a negative modulation of the
ATPase activity. The absence of these clusters in the amino-terminal
truncated protein or their neutralization by polyanions prevents the
negative modulation. Polyanions also stimulate the ATPase activity of
the amino-terminal truncated protein, suggesting a regulatory
functionality for other domains of the molecule.
Figure 6:
Kinetic
parameters of complete and amino-terminal truncated Yhs4p ATPase.
ATPase activity was measured for 10 min as a function of four ATP
concentrations in the range from 5 to 100 µ
M as described
under ``Experimental Procedures.'' Vand K values were calculated by Lineweaver-Burk plot.
Each point represents the mean of three determinations assayed in
duplicate. Number in parentheses is percent of
inhibition. K229Q point mutation in Yhs4p and was obtained as described
under ``Experimental Procedures.'' n.d. indicates
``not determined.''
Figure 7:
Effect of several polyanions and
polylysine on the ATPase activity of complete and amino-terminal
truncated Yhs4p. ATPase activity was assayed as described under
``Experimental Procedures.'' Each point represents the
average of three determinations. Polyanions and polylysine were added
to a final concentration of 10 µg/ml. The ATPase activity was
assayed without additions ( closed circles) and after the
addition of polylysine ( circles) or single-stranded DNA
( inverted triangles), double-stranded DNA
( triangles), RNA ( diamonds), or poly(Glu-Ala-Tyr)
( squares).
The nucleotide
specificity of the Yhs4p, examined by testing the effect of unlabeled
nucleotides on the hydrolysis of [-
P]ATP,
showed that GTP, UTP, and CTP compete with
[
-
P]ATP, resulting in a significant
inhibition of the ATPase activity (Fig. 8). Therefore, Yhs4p
interacts with all four nucleotides as predicted by the domains found
in its sequence (Fig. 1).
Figure 8:
Nucleoside triphosphate specificity of
complete and amino-terminal truncated Yhs4p ATPase. ATPase activity was
assayed for 15 min as described under ``Experimental
Procedures.'' Cold nucleoside triphosphate was added to the ATPase
assay medium at a final concentration of 500 µ
M. Each
bar represents the average of three
determinations.
73%) in the whole span of their primary sequence with a lower
identity (
50%) in the first 40 amino acid residues of their amino
termini. The divergence observed between Yhs4p and other members of
this subfamily is greatest at their amino-terminal region. For instance
Yhs4p and CIM5, a S. cerevisiae protein proposed to be
involved in 26 S protease activity in vivo (21) , share
only 4% identity in the first 180 residues from their amino-terminal
region (Fig. 2, central diagram). A distinct
feature of the Yhs4p amino-terminal is the presence of two clusters of
positively charged amino acid residues (Fig. 1). The
amino-terminal truncated Yhs4p has a V
3 times
higher than the complete molecule (Fig. 6). To prove the
hypothesis that the clusters of positive amino acid residues confer a
negative modulation on the ATPase activity of Yhs4p we studied the
effect of several polyanions and two polycations on the activity of
amino-terminal truncated and complete Yhs4p. The stimulation by
polyanions was overall 3-fold higher in the complete molecule than in
the amino-terminal truncated molecule, while polylysine inhibited only
the activity of amino-terminal truncated Yhs4p by 48% (Fig. 7).
These results support the idea that the positive residues in the
amino-terminal region result in it functioning as an internal modulator
of Yhs4p ATPase activity. The activation of Yhs4p by polyanions appears
to be unspecific, suggesting a putative mechanism for in vivo modulation of the ATPase through the interaction with negatively
charged epitopes of neighboring proteins. Yhs4p as part of a
macromolecular complex may become activated upon interaction with the
proper subunit during the assembly of the 26 S protease complex.
Regarding this possibility, it is interesting to note that some
subunits in the 20 S proteasome and in the activator PA-700 contain a
cluster of three or more acidic residues at their carboxyl termini as
for instance in the 20 S proteasome subunits HC8
(
EEDESDDD
) and HC9
(
EEEE
) in human
(35) ; Y13
(
EDEEADED
)
(36) , YC1
(
DDEEDEDD
)
(37) , and PRE2
(
DDD
)
(38) in yeast; XC8
(
EEEDDSDDD
) and XC9-1
(
EEEE
)
(39) in Xenopus;
and p58 (
EDDDD
)
(8) in the PA700
activator.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.