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Vol. 13, Issue 9, 3005-3028, September 2002

andDepartment of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202
Submitted April 12, 2002; Revised May 28, 2002; Accepted June 5, 2002| |
ABSTRACT |
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Saccharomyces cerevisiae Pkh1 and Pkh2 are
functionally redundant homologs of mammalian protein kinase,
phosphoinositide-dependent protein kinase-1. They activate two
closely related, functionally redundant enzymes, Ypk1 and Ykr2
(homologs of mammalian protein kinase, serum- and
glucocorticoid-inducible protein kinase). We found that Ypk1 has a more
prominent role than Ykr2 in mediating their shared essential function.
Considerable evidence demonstrated that Pkh1 preferentially activates
Ypk1, whereas Pkh2 preferentially activates Ykr2. Loss of Pkh1 (but not
Pkh2) reduced Ypk1 activity; conversely, Pkh1 overexpression increased
Ypk1 activity more than Pkh2 overexpression. Loss of Pkh2 reduced Ykr2
activity; correspondingly, Pkh2 overexpression increased Ykr2 activity
more than Pkh1 overexpression. When overexpressed, a catalytically
active C-terminal fragment (kinase domain) of Ypk1 was growth
inhibitory; loss of Pkh1 (but not Pkh2) alleviated toxicity. Loss of
Pkh2 (but not Pkh1) exacerbated the slow growth phenotype of a
ypk1
strain. This Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy
is not absolute because all double mutants (pkh1
ypk1
, pkh2
ypk1
, pkh1
ykr2
, and pkh2
ykr2
) were viable.
Compartmentation contributes to selectivity because Pkh1 and Ypk1 were
located exclusively in the cytosol, whereas Pkh2 and Ykr2 entered the
nucleus. At restrictive temperature,
ypk1-1ts ykr2
cells
lysed rapidly, but not in medium containing osmotic support. Dosage and
extragenic suppressors were selected. Overexpression of Exg1 (major
exoglucanase), or loss of Kex2 (endoprotease involved in Exg1
processing), rescued growth at high temperature. Viability was also
maintained by PKC1 overexpression or an activated allele of the downstream protein kinase (BCK1-20). Conversely,
absence of Mpk1 (distal mitogen-activated protein kinase of the
PKC1 pathway) was lethal in
ypk1-1ts ykr2
cells.
Thus, Pkh1-Ypk1 and Pkh2-Ykr2 function in a novel pathway for cell wall
integrity that acts in parallel with the Pkc1-dependent pathway.
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INTRODUCTION |
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A cascade of protein kinases is a commonly used mechanism for
amplifying and disseminating signals that control metabolism, growth,
survival, and differentiation in eukaryotic cells. In animal cells,
recruitment of phosphatidylinositol 3-kinase by growth factor
receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1) (for review, see
Toker and Newton, 2000
; Vanhaesebroeck and Alessi, 2000
). Activated PDK1 phosphorylates and activates multiple downstream targets, including protein kinase B/c-Akt (Brazil and Hemmings, 2001
; Lawlor and Alessi, 2001
), p70 S6 kinase (Alessi et
al., 1998
; Kozma and Thomas, 2002
), protein kinase C (PKC)
isoforms (Chou et al., 1998
; Le Good et al.,
1998
), and serum- and glucocorticoid-inducible protein kinase (SGK)
isoforms (Kobayashi and Cohen, 1999
; Kobayashi et al.,
1999
), thereby eliciting physiological responses.
We have demonstrated previously that, in budding yeast
(Saccharomyces cerevisiae), Pkh1 and Pkh2 are the homologs
and functional equivalents of mammalian PDK1. Pkh1 and Pkh2 share an
essential function because pkh1
and pkh2
single mutants are viable, whereas a pkh1
pkh2
double
mutant is inviable. Expression of human PDK1 rescues the lethality of a
pkh1
pkh2
strain (Casamayor et al., 1999
).
The PDK1 enzymes from Caenorhabditis elegans,
Drosophila melanogaster, and Homo sapiens all
possess a C-terminal pleckstrin homology (PH) domain that binds
phosphatidylinositol (PtdIns)(3,4,5)P3 and PtdIns(3,4)P2 (Stephens et al.,
1998
; Currie et al., 1999
; Fruman et al., 1999
).
However, S. cerevisiae does not produce PtdIns(3,4,5)P3 or
PtdIns(3,4)P2 (Hawkins et al., 1993
;
De Camilli et al., 1996
). Moreover, Pkh1 and Pkh2 lack
discernible PH domains, and PDK1 lacking its PH domain was sufficient
to rescue the growth of pkh1
pkh2
cells (Casamayor
et al., 1999
), suggesting that the activity of Pkh1 and Pkh2
in yeast does not depend on phosphoinositides. It was shown
subsequently that sphingosine (4-dehydro-sphinganine) can also
stimulate mammalian PDK1 autophosphorylation and increase its ability
to phosphorylate in vitro known PDK1 substrates, such as c-Akt and
PKC
(King et al., 2000
). Correspondingly, it has been
reported recently that Pkh1 and Pkh2 can be activated in vitro by
nanomolar concentrations of the major sphingoid base in yeast,
phytosphingosine (4-hydroxy-sphinganine) (Friant et al.,
2001
). Moreover, endocytosis in yeast seems to require sphingoid base
synthesis and overexpression of Pkh1 or Pkh2 can suppress this
requirement (Friant et al., 2001
), suggesting that sphingoid bases activate a signaling pathway involving Pkh1 and Pkh2.
Mammalian PDK1 activates its downstream targets by phosphorylating a
Thr residue (starred) in a sequence motif,
Thr*-Phe-Cys-Gly-Thr-X-Glu-Tyr (where X represents any amino acid),
that lies within the "activation loop" of their catalytic domains
(Hanks and Hunter, 1995
) and is unique to and conserved in all known
PDK1 substrates. Full activation of c-Akt/PKB and other PDK1 targets
also seems to require phosphorylation at a second site (starred)
situated in a hydrophobic motif, Phe-X-X-Ar-Ser*/Thr*-Ar (where Ar
represents an aromatic residue), that is located near the C terminus of
each of these enzymes (Toker and Newton, 2000
; Vanhaesebroeck and
Alessi, 2000
). In S. cerevisiae, four previously
characterized protein kinases possess both of these motifs, suggesting
that they are physiological substrates of Pkh1 and/or Pkh2. These four
protein kinases are the products of the following genes:
YPK1 (Maurer, 1988
), YKR2/YPK2 (Maurer, 1988
;
Chen et al., 1993
), PKC1 (Levin et
al., 1990
), and SCH9 (Toda et al., 1988
).
Studies from this laboratory have demonstrated that Ypk1 is a direct
substrate of Pkh1 (Casamayor et al., 1999
) and that Ykr2 is
phosphorylated by Pkh2 (Torrance, 2000
). Similarly, it has been shown
that Pkc1 can also be phosphorylated by Pkh1 and Pkh2 (Inagaki et
al., 1999
; Friant et al., 2001
). Reduced Pkc1 activity
was observed in a pkh1-1ts
pkh2
strain, and the temperature sensitivity of this
strain was partially suppressed by a dominant PKC1(R398P)
allele, suggesting that Pkh1 and Pkh2 are required for Pkc1 function in
vivo (Inagaki et al., 1999
).
The catalytic domains of Ypk1 and Ykr2 are 88% identical and these
proteins also share extensive homology across their N- and C-terminal
extensions. Moreover, the catalytic domains of Ypk1 and Ykr2 closely
resemble (55% identity) that of mammalian SGK. Indeed, cells lacking
Ypk1 or Ykr2 are viable, whereas cells lacking both Ypk1 and Ykr2 are
inviable (Chen et al., 1993
; Schnieders, 1996
), and
expression of mammalian SGK rescues this inviability (Casamayor
et al., 1999
). Furthermore, both purified PDK1 and purified
Pkh1 phosphorylate the same residue (Thr504) in the consensus motif in
purified Ypk1, and Ypk1 phosphorylation is significantly diminished in
vivo in cells lacking Pkh1 (Casamayor et al., 1999
). Thus,
just as SGK is a downstream target of PDK1 in animal cells, Ypk1 and
Ykr2 seem to act downstream of Pkh1 and Pkh2 in yeast. Moreover,
lipid-derived signals are required as upstream activators in both
pathways, 3-phosphoinositides and sphingosine in the case of PDK1 and
closely related sphingoid bases in the case of Pkh1 and Pkh2.
Consistent with this view, overexpression of Ypk1 confers resistance to
myriocin (ISP-1), an antibiotic that specifically inhibits serine
C-palmitoyltransferase (product of the LCB1 gene), which is
the enzyme responsible for sphinganine biosynthesis (Sun et
al., 2000
).
Herein, we describe experiments that address the genetic and
biochemical interrelationships between Pkh1 and Pkh2 and Ypk1 and Ykr2,
which we undertook to try to understand the reason for the redundancies
within these protein kinase cascades. To provide further insight, we
also investigated the subcellular localization of all four proteins.
Finally, as two independent approaches for discerning the physiological
function of the Ypk1 and Ykr2 enzymes, we selected for dosage
suppressors and also for chromosomal mutations that suppress the lysis
phenotype of ypk1-1ts ykr2
cells.
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MATERIALS AND METHODS |
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Strains and Growth Conditions
Yeast strains used in this study are listed in Table
1. Standard rich (YP) and defined minimal
(SC) media (Sherman et al., 1986
), containing either 2%
glucose (Glc), 2% raffinose (Raf), or 2% galactose (Gal) as the
carbon source and supplemented with appropriate nutrients to maintain
selection for plasmids, were used for yeast cultivation. For gene
expression from the galactose-inducible GAL1 promoter in
liquid media, cells were pregrown to mid-exponential phase in SC
containing 2% raffinose-0.2% sucrose (Raf/Suc) and then Gal was added
to a final concentration of 2% and incubation continued for 2 h.
In experiments involving growth on solid medium containing
5-fluoroorotic acid, 5-fluoroorotic acid was used at a concentration of
0.5 mg/ml (Boeke et al., 1984
). Cells were grown routinely
at 30°C, except for strains carrying temperature-sensitive mutations,
which were propagated at their permissive temperature (26°C).
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Recombinant DNA Methods
Escherichia coli strain DH5
(Hanahan, 1983
) was
used for the construction and propagation of plasmids. Conventional
recombinant DNA methods were used for the construction of plasmids
(Sambrook et al., 1989
). The sequences of constructs that
contained DNA fragments amplified by polymerase chain reaction (PCR)
were verified by the dideoxy chain termination-sequencing method
(Sanger et al., 1977
). Native and Turbo Pfu polymerases
(Stratagene, La Jolla, CA) were used for PCR, unless noted otherwise.
Plasmids
Plasmids pYPK1, pYKR2,
pGAL-YPK1, pGAL-YKR2 (pAM1),
pGAL-Ypk1-Myc (pAM54), pADH-YPK1,
pRS316-YKR2 (pAM12), pGAL-PKH1
(pAM73), and pGAL-PKH2 (pAM79) have been
described previously (Maurer, 1988
; Kubo et al., 1989
;
Casamayor et al., 1999
). To create plasmid pADH-YKR2 (pAM4), which constitutively
overexpresses YKR2 from the ADH1 promoter, a
2.4-kb XhoI (blunt)-SalI fragment containing the
entire YKR2 gene was excised from pYKR2, gel
purified, and inserted into vector pAD4 M (Martin et al.,
1990
) that had been linearized with SmaI/SalI. To
generate a version of Ykr2 tagged at its C-terminal end with the c-Myc
epitope (Evans et al., 1985
), a PCR-based method for precise
gene fusion (Yon and Fried, 1989
) was performed using the
YKR2 sequence cloned in pUC18 as one template (pYKR2), and as the other template, pOGFP (E. Swartzman,
this laboratory), which contains a sequence encoding the 16-residue version of the c-Myc epitope followed by a (His)6
tag cloned in pBluescript (Stratagene); with three appropriate
synthetic oligonucleotide primers: T3 (Stratagene); 5'-GGA CAT ATT GCA
CTG TGT G-3' (RMN5), corresponding to sequences in YKR2
overlapping a DraIII site near the C terminus; and a
"joiner" primer, 5'-TTC AGA AAT CAA CTT TTG TTC ACT AAT GCT TCT CCC
CTG-3' (RMC), corresponding to the 3' end of the YKR2 coding
sequence and the first several residues of the c-Myc epitope. An
~1.6-kb DraIII/KpnI fragment of the resulting PCR product was used to replace the corresponding segment in
pYKR2, yielding pYkr2-Myc (pAM24). An ~3-kb
NcoI/HindIII fragment from pYkr2-Myc was gel
purified and used to replace the corresponding ~2.2-kb
NcoI/HindIII segment in
pGAL-YKR2 to create a 2-µm DNA-containing, LEU2-marked plasmid, pGAL-Ykr2-Myc (pAM59), that
overexpresses Ykr2-Myc upon galactose induction. To generate a
catalytically inactive ("kinase-dead") version of Ypk1, a PCR-based
method for site-directed mutagenesis was performed using
pYPK1 as the template and three appropriate synthetic
oligonucleotide primers: 5'-CTT GAA CAC AGT AAG TAA CGG-3' (PKC2),
corresponding to the flanking genomic sequence commencing 68-base pairs
downstream of the stop codon; 5'-CAC AAA AAG TAT ACG CCT
TGG CGG CAA TCA G-3' (PKD), where the underlined nucleotide
is a silent mutation to introduce a BglI site, and the bold
nucleotides correspond to an introduced alanine codon (GCG) in place of
the native lysine codon (AAG); and 5'-GTC CAT CGA TGA TTT CGA TC-3'
(Pseq2), corresponding to the coding strand of YPK1 starting
at nucleotide position 1024. The resulting ~1.1-kb PCR product was
digested with ClaI and NcoI, and the resulting
~850-base pair fragment was used to replace the corresponding segment
in pYPK1, yielding pYPK1(K376A-KD) (pAM46). Conversion of the Lys residue at the equivalent position in all other
protein kinases examined to date eliminates their catalytic activity
(Hanks and Hunter, 1995
). To generate a catalytically inactive
(kinase-dead) version of Ykr2, a similar PCR-based approach for
site-directed mutagenesis was performed using pYKR2 as the template, and three appropriate synthetic oligonucleotide primers: 5'-AGT ATA GCC CTG CCC CAA C-3' (Rseq2), corresponding to the noncoding
strand of YKR2 commencing at nucleotide position 1544; 5'-CCC AAA AGA TTT ACG CCT TGG CGG CTC TGA G-3'
(RKD), where the underlined nucleotide is a silent mutation to
introduce a BglI site, and the bold nucleotides correspond
to an introduced alanine codon (GCG) in place of the native lysine
codon (AAG); and 5'-CGT GGG GTA ATG GCC TG-3' (Rseq3), corresponding to
the coding strand of YKR2 starting at nucleotide position
66. The resulting ~1.4-kb PCR product was digested with
NcoI and DraIII and used to replace the
corresponding segment in pYKR2, yielding pYKR2(K373A-KD) (pAM47). Plasmid pYPK1(K376A-KD)
was digested with AlwnI, converted to flush ends by
treatment with T4 polymerase (NEB) and all four dNTPs then digested
with SalI. The resulting 3.3-kb
YPK1(K376A-KD)-containing fragment was gel purified and ligated into YEp351GAL that had been linearized by digestion
with XbaI, converted to flush ends by incubation with T4
polymerase and all four dNTPs, and then digested with
SalI. The resulting plasmid,
pGAL-YPK1(K376A-KD) (pAM48), expresses a
catalytically inactive allele [Ypk1-(K376A-KD)] from a 2-µm
DNA-containing, LEU2-marked plasmid under control of the
GAL1 promoter. An ~1.2-kb NcoI/SalI
fragment from pYpk1-Myc was gel purified and used to replace the
corresponding NcoI/SalI segment in
pGAL-YPK1(K376A-KD) to create
pGAL-YPK1(K376A-KD)-Myc (pAM49). An ~2.4-kb
XhoI-HindIII fragment containing the entire
YKR2(K373A-KD) allele was excised from
pYKR2(K373A-KD), gel purified, and inserted into
YEp351GAL that had been linearized with SalI and
HindIII, to create a 2-µm DNA-containing,
LEU2-marked plasmid,
pGAL-YKR2(K373A-KD) (pAM50), that overexpresses
catalytically inactive Ykr2 upon galactose induction.
Galactose-inducible expression vectors that are URA3 based
were constructed as follows. An ~3.3-kb
BamHI/HindIII fragment carrying YPK1
was excised from pGAL-YPK1, gel purified, and
ligated into YEp352GAL (Benton et al., 1994
),
which had been linearized with BamHI/HindIII,
yielding YEp352GAL-YPK1 (pAM75). An ~3.8-kb BamHI/HindIII fragment from
pGAL-Ypk1-Myc was gel purified and ligated into
YEp352GAL that had been linearized with
BamHI/HindIII, yielding
YEp352GAL-Ypk1-Myc (pAM76). An ~2.2-kb
BamHI/HindIII fragment from
p2GAL-YKR2 was gel purified and ligated into
YEp352GAL, which had been linearized with
BamHI/HindIII, yielding
YEp352GAL-YKR2 (pAM77). An ~3.0-kb
BamHI/HindIII fragment from
p2GAL-Ykr2-Myc was gel purified and ligated into
YEp352GAL, which had been linearized with
BamHI/HindIII, yielding
YEp352GAL-Ykr2-Myc (pAM78). To generate an amino-terminal
truncation of Ypk1, the following two-step approach was taken. First,
an ~1.1-kb fragment corresponding to the last 344 amino acids of Ypk1
was amplified by PCR from pYPK1 with the following
oligonucleotides: 5'-GGC GGA TCC ATG TCC AGA AAT
AAA CCT TTG TCC-3' (PCT), corresponding to sequences in the middle of
the YPK1 coding sequence, just upstream of the beginning of
the catalytic domain, where the underlined nucleotides correspond to an
introduced BamHI restriction site, and the bold nucleotides represent an introduced start codon (ATG); and 5'-CTT GAA CAC AGT AAG
TAA CGG-3' (PKC2), corresponding to the flanking genomic sequence
commencing 68-base pairs downstream of the stop codon. The resulting
PCR product was digested with BamHI and NcoI, gel purified, and used to replace an ~2.6-kb
BamHI/NcoI fragment in pRS315-YPK1(B/H). The resulting CEN-containing,
LEU2-marked plasmid encodes an amino-terminal truncation of
Ypk1, which contains only the catalytic domain, but essentially no
promoter sequence, and is called pRS315-YPK1-
N (pAM55).
An ~1.2-kb NcoI/SalI fragment from
pRS315-Ypk1-myc was gel purified and used to replace the corresponding
NcoI/SalI segment in pRS315-YPK1-
N
to create a CEN-containing, LEU2-marked plasmid,
pRS315-Ypk1-
N-myc (pAM56), that encodes a myc-tagged version of the
Ypk1 catalytic domain. To insert a promoter, an ~2.3-kb
BamHI/SalI fragment from
pRS315-YPK1-
N was gel purified and inserted into
YEp351GAL that had been linearized with
BamHI/SalI to create a 2-µm
NA-containing,
LEU2-marked plasmid, pGAL-YPK1-
N
(pAM99), that overexpresses the amino-terminal truncation of Ypk1 upon
galactose induction. Likewise, an ~2.0-kb
BamHI/SalI fragment from pRS315-Ypk1-
N-Myc was
gel purified and inserted into YEp351GAL that had been
linearized with BamHI/SalI to create a 2-µm
NA-containing, LEU2-marked plasmid,
pGAL-Ypk1-
N-Myc (pAM100), that overexpresses a myc-tagged
version of the amino-terminal truncation of Ypk1 upon galactose
induction. To move these truncated Ypk1 derivatives into
URA3-marked plasmids, an ~2.3-kb
BamHI/SalI fragment from
pGAL-YPK1-
N was gel purified and inserted into YEp352GAL that had been linearized with
BamHI/SalI to create a 2-µm DNA-containing,
URA3-marked plasmid,
YEp352GAL-YPK1-
N (pAM101), that overexpresses
the amino-terminal truncation of Ypk1 upon galactose induction.
Similarly, an ~2.0-kb BamHI/SalI fragment from
pGAL-Ypk1-
N-Myc was gel purified and inserted into
YEp352GAL that had been linearized with
BamHI/SalI to create a 2-µm DNA-containing, URA3-marked plasmid, YEp352GAL-Ypk1-
N-myc
(pAM102), that overexpresses a myc-tagged version of the amino-terminal
truncation of Ypk1 upon galactose induction. To generate a
catalytically inactive derivative of the Ypk1-
N allele, a 2.3-kb
ClaI/HindIII fragment from
pGAL-YPK1-KD, encoding the carboxy terminus (containing the K376A kinase-dead mutation of Ypk1) was gel purified and used to
replace the corresponding segment in
YEp352GAL-Ypk1-
N-Myc. The resulting plasmid,
YEp352GAL-Ypk1-
N-KD (pFR30), overexpresses a
catalytically inactive derivative of the amino-terminal truncation of
Ypk1 upon galactose induction.
Protein Localization by Using Chimeras Containing Green Fluorescent Protein (GFP)
To create vectors for galactose-inducible expression of
YPK1, YKR2, and PKH2, each fused to
the carboxy terminus of a protein comprising three tandem repeats of an
enhanced (S65T V163A) mutant of GFP, the following approach was taken.
Two primers, 5'-GCG AGC GGG ATC CAT G, the first
18 bases of the gene-3' (primer A), where underlined bases correspond
to an introduced BamHI site and start codon in bold; and
5'-GGC ACG CGT CGA CTT A, the last 18 bases of
the gene-3' (primer B), where underlined bases correspond to an
introduced SalI site and stop codon in bold, were used to
amplify the entire open reading frames of the corresponding genes from
genomic DNA. The PCR products were digested with BamHI and
SalI and ligated into vector pGS836 (YCpGAL-3GFP) (Maurer et al., 2001
) that had been digested with
BamHI and SalI, yielding plasmids
pGAL-3GFP-YPK1 (pFR33),
pGAL-3GFP-YKR2 (pER2), and
pGAL-3GFP-PKH2 (pER3). To create
pGAL-3GFP-PKH1 (pFR37), the same approach was
used, but due to the presence of BamHI and SalI restriction sites in the gene, two PCR products were made, one with
primer A and a primer corresponding to the sequence 3' to the
ClaI site present in PKH1, and the other with a
primer 3' of the ClaI site and primer B. A three-way
ligation was then used to ligate the two PCR products digested with
BamHI and ClaI, or ClaI and
SalI, into pGS836 that had been digested with
BamHI and SalI.
Cells expressing the GFP constructs were grown to mid-exponential phase at 30°C in SC-Leu containing Raf/Suc and then induced with 2% galactose for 3 h. Nuclear DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) by adding the dye directly in the medium at a concentration of 1 µg/ml for the last hour of growth. Rhodamine-labeled phalloidin was purchased from Molecular Probes (Eugene, OR). Samples of each culture were viewed directly with a TE300 fluorescence microscope (Nikon, Melville, NY) equipped with a 100×/1.4 Plan-Apo objective and a 1.4 numerical aperture condenser. Digital images were acquired with a bottom-ported Orca 100 charge-coupled device camera (Hamamatsu, Bridgewater, NJ) and Phase 3 Imaging Systems software (Northern Exposure, Inc., Glen Mills, PA). The fraction of lysed cells in cultures was assessed by direct counting of at least 200 cells after staining with a vital dye (LIVE/DEAD Yeast Viability kit; catalog no. 7009; Molecular Probes).
Antibody Production
To express a glutathione S-transferase (GST)-Ypk1 fusion protein in E. coli, an ~370-base pair fragment corresponding to the first 114 amino acids of Ypk1 was amplified by PCR from pYPK1 by using the following oligonucleotides: 5'-GGG GGG GGA TCC ATG TAT TCT TGG AAG TCA AAG TTT-3', where the underlined nucleotides correspond to an introduced BamHI site, and the bold nucleotides correspond to the start codon; and 5'-GGG GGG AAT TCT CAG GTG GCA TCA TTG GGT GTC CC-3', where the underlined nucleotides correspond to an introduced EcoRI site, and the bold nucleotides correspond to the reverse complement of an introduced stop codon. This PCR fragment was then digested with BamHI and EcoRI, gel purified, and ligated into the pGEX2T vector (Pharmacia, Peapack, NJ), which had been linearized with BamHI and EcoRI to create plasmid pGEX-Ypk1 (pAM5). To express a GST-Ykr2 fusion protein in E. coli, an ~360-base pair fragment corresponding to the first 111 amino acids of Ykr2 was amplified by PCR from pYKR2 by using the following olignucleotides: 5'-GGG GGG GGA TCC ATG CAT TCC TGG CGA ATA TCC AAG-3', where the underlined nucleotides correspond to an introduced BamHI site, and the bold nucleotides correspond to the start codon; and 5'-GGG GGG AAT TCT CAA CTC GGT CCC TGC GTC TCA GT-3', where the underlined nucleotides correspond to an introduced EcoRI site, and the bold nucleotides correspond to the reverse complement of an introduced stop codon. This PCR fragment was then digested with BamHI and EcoRI, gel purified, and ligated into the pGEX2T vector, which had been linearized with BamHI and EcoRI to create plasmid pGEX-Ykr2 (pAM6).
To prepare antigen, expression of GST-Ypk1(1-114) and GST-Ykr2(1-111)
fusions from plasmids, pGEX-Ypk1 and pGEX-Ykr2, respectively, were
induced in a protease-deficient E. coli strain BL21
(DE3)[pLys] (Studier, 1991
) by addition of
isopropyl-
-D-thiogalacto-pyranoside to a final
concentration of 0.2 mM followed by incubation with aeration for 2 h at 30°C. Cells were harvested, washed once with ice-cold wash
buffer (50 mM Tris-HCl pH 8, 0.5 mM dithiothreitol [DTT], 100 mM KCl,
1 mM phenylmethylsulfonyl fluoride, 0.05% NP-40, 1 mM EGTA, and 1 mM
EDTA), and resuspended in 1/20 volume of wash buffer containing 1 M
NaCl. Cells were disrupted by digestion with lysozyme (final
concentration, 2 mg/ml) followed by sonication. Insoluble material was
removed by centrifugation at 12,000 × g, and the
soluble GST-Ypk1(1-114) or GST-Ykr2(1-111) proteins were purified by
adsorption to, and elution from, glutathione-agarose beads (Pharmacia),
essentially as directed by the manufacturer, except that elution was
performed in the presence of 1 M NaCl and 20 mM glutathione. The
purified proteins were used as immunogens to raise polyclonal antisera
in adult female New Zealand White rabbits following standard
immunization protocols (Harlow and Lane, 1988
). The resulting anti-Ypk1
antibodies (serum #1446) and anti-Ykr2 antibodies (serum #1732) are
specific to Ypk1 and Ykr2 and do not display any cross-reaction against
the incorrect antigen. Anti-GFP antibodies were the generous gift of
Roger Tsien and Charles Zuker (Department of Cellular and
Molecular Medicine, University of California, San Diego, CA).
Preparation of Cell Extracts and Immunoblot Analysis
Yeast cells were grown at 30°C to mid-exponential phase
(A600 nm = 0.5-1), either in SC medium
supplemented in a manner appropriate for maintenance of plasmids or in
rich medium (YPGlc). If cells required galactose induction for
expression from the GAL1 promoter, galactose was added to a
final concentration of 2% and the cultures were incubated at 30°C
for an additional 2 h. Cells were harvested by brief
centrifugation, washed twice by resuspension and resedimentation in
ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 3 mM DTT and 1 mM phenylmethylsulfonyl fluoride), and resuspended in 200 µl of the
same buffer. Prechilled glass beads (0.45-0.6 mm in diameter) were
added to the meniscus of the cell suspension, and lysis was achieved by
vigorous vortex mixing for six 1-min intervals, with intermittent
cooling on ice. To remove the glass beads, the bottom of the Eppendorf
tube was punctured with a syringe needle (<0.5 mm in diameter) and
inserted into another tube; the lysate was collected into the fresh
tube by brief centrifugation in a clinical centrifuge. The crude
extract was subjected to centrifugation at 30,000 × g
for 15 min to remove unbroken cells and large debris. The protein
concentration of the crude extract was measured using a dye-binding
method (Bradford, 1976
) with a protein assay kit as instructed by the
manufacturer (Bio-Rad, Hercules, CA), by using bovine serum albumin
(New England Biolabs, Beverly, MA) as the standard.
For immunoblot analysis, samples (50 µg of total protein)
were diluted into SDS-PAGE sample buffer (Laemmli, 1970
), subjected to
electrophoresis in an 8-12% gel, and then transferred to
nitrocellulose (Towbin et al., 1979
). To detect Ypk1, rabbit
polyclonal anti-Ypk1 antiserum #1446 was used at a dilution of 1:3000.
To detect Ykr2, rabbit polyclonal anti-Ykr2 antiserum #1732 was used at
a dilution of 1:3000. To detect proteins using the anti-c-Myc
monoclonal antibody (mAb) 9E10, ascites fluid containing this mAb was
used at a dilution of 1:10,000 (Evans et al., 1985
).
Immobilized immune complexes were detected using a commercial
chemiluminescence detection system (Renaissance; PerkinElmer Life
Sciences, Boston, MA) and x-ray film (Biomax MR; Eastman Kodak,
Rochester, NY).
Immunoprecipitations
Yeast cultures to be used for immunoprecipitation analysis were grown as described above, and then rinsed in ice-cold IP buffer (20 mM Tris-HCl pH 7.5, 125 mM potassium acetate, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 0.1% Triton X-100, and 12.5% glycerol). Glass beads were added to the meniscus of the cell suspension, and lysis was achieved by vigorous vortex mixing for eight 30-s intervals with intermittent cooling on ice. The lysate was clarified by centrifugation at 14,000 × g at 4°C for 30 min. The clarified extract was assayed for protein concentration, and a sample (1 mg of total protein) was diluted to a final volume of 200 µl in IP buffer. An aliquot (20 µl) of protein G/protein A-agarose beads (30% slurry) (Oncogene Science, Cambridge, MA) and a sample of an appropriate control antibody, either 2 µl of preimmune rabbit serum or 1 µg of purified mouse anti-T-cell receptor antibody (gift of James Allison, Department of Molecular and Cell Biology, University of California, Berkeley, CA), were added. The samples were then incubated on a roller drum for 1 h at 4°C to adsorb proteins that bound nonspecifically to the solid support and to rabbit or mouse IgG (preclearing). The beads were removed by centrifugation for 10 min in a microfuge, and the supernatant fraction was transferred to a fresh tube containing another aliquot (15 µl) of protein G/protein A-agarose beads and either 2 µl of anti-Ypk1 (or anti-Ykr2) polyclonal antiserum or 1 µl of anti-c-Myc (mAb 9E10) ascites, and incubated on a roller drum for 1 to 3 h at 4°C. The beads were sedimented by brief centrifugation in a microfuge and washed three times (1 ml each) with ice-cold IP buffer and collected by centrifugation for 1 min in a microfuge on maximum speed. Bead-bound immune complexes were solubilized in SDS-PAGE sample buffer and immediately boiled for 5 min in a water bath and then clarified by brief centrifugation in a Microfuge before resolution by SDS-PAGE. The proteins of interest were visualized as described above.
Immune-Complex Protein Kinase Assays
Cells expressing either wild-type or kinase-dead Ypk1-myc (or
Ykr2-myc) under control of the GAL1 promoter were grown in
SC containing Raf/Suc to an A600 nm = 0.6, induced by addition of galactose (2% final concentration), incubated
with shaking at 30°C for 2 h, collected by centrifugation,
washed with ice-cold 1× phosphate-buffered saline, resuspended in 0.2 ml of ice-cold IP buffer, and lysed as described above. The resulting
lysates were clarified by centrifugation at 4°C for 30 min at
30,000 × g. Protein concentration in the resulting
crude extracts was determined by the Bradford (1976)
method. A
volume of extract containing 1 mg of total protein was
immunoprecipitated with mAb 9E10 as described above. The
immunoprecipitates were washed once with ice-cold IP buffer, once with
ice-cold IP buffer containing 0.5 M NaCl, and twice with ice-cold
buffer A (50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, and 0.1% [by vol]
2-mercaptoethanol). As part of the final wash, the slurry of beads was
split into two equal portions. For immunoblot analysis,
SDS-PAGE sample buffer (~15 µl) was added directly to one sample of
each bead suspension. For protein kinase assays, the activity of the
Ypk1-myc or Ykr2-myc immune complex was assayed by adding 30 µl of a
mixture containing 1 µM microcystin-LR, 10 mM Mg-acetate, 100 µM
[
-32P]ATP (200-400 cpm/pmol), and 100 µM
Cross-tide (GRPRTSSFAEG) (Cross et al., 1995
), which we have
documented previously is an excellent peptide phospho-acceptor
substrate for Ypk1 and Ykr2 (Casamayor et al., 1999
;
Torrance, 2000
). After incubation for 15 min at 30°C, each reaction
was terminated by spotting a portion (45 µl) of the reaction mixture
onto small squares of phosphocellulose paper (P81; Whatman, Maidstone,
United Kingdom), which were washed and analyzed as described in detail
previously (Alessi et al., 1995
). In some experiments,
samples of the immunoprecipitates were resuspended in an appropriate
buffer (20 mM Tris-HCl pH 8.8 and 10 mM MgCl2)
and treated with shrimp alkaline phosphatase (0.25 U; US Biochemical,
Cleveland, OH) in either the absence or presence of a mixture of
inhibitors of this phosphatase (25 µM Na-orthovanadate and 100 µM
-glycerol-phosphate, adjusted to pH 8).
Bioassays for Drug Sensitivity
An agar diffusion (halo) assay (Reneke et al., 1988
)
was performed to test the relative sensitivity of various strains to rapamycin, valinomycin, hygromycin B, cycloheximide, and polyoxin D. Nascent lawns of the strains to be tested were prepared by mixing
~2 × 106 cells from a saturated culture
with 2 ml of molten (55°C) 1% agar. The cell-containing agar was
rapidly mixed and immediately poured evenly onto plates containing an
appropriate medium. Various concentrations of rapamycin (50 and 500 ng/µl), hygromycin B (5 and 50 µg/µl), or the other drugs
indicated, were spotted in the same volume (10 µl) onto sterile
cellulose filter discs (0.6 cm), which were placed on the nascent lawn.
The plates were incubated at 30°C, and photographed after 2 d.
Selection and Analysis of Dosage Suppressors
A library of restriction fragments of yeast genomic DNA cloned
into a URA3-marked, 2-µm DNA-based vector, YEp352 (Hill
et al., 1986
), was introduced into strain YPT40
(ypk1-1ts ykr2
) (Casamayor
et al., 1999
) by selecting transformants on SCGlc-Ura medium
at 26°C. Temperature-resistant clones were then selected by their
ability to grow at 35°C. One suppressor plasmid obtained carried the
EXG1 locus as the sole open reading frame. To create a
plasmid that expressed EXG1 from a high-level constitutive promoter, two primers, 5'-GCG TCT CGA GAT GCT
TTC GCT TAA AA-3', where the underlined bases correspond to an
introduced XhoI site, and the start codon is in bold; and
5'-CGC CGG AGC TC T TAG TTA GAA ATT GTG CC-3',
where the underlined bases correspond to an introduced SacI
site, and the stop codon is in bold, were used to amplify the entire
open reading frame of EXG1 from the library plasmid that was
originally isolated as a dosage suppressor of the
ypk1-1ts ykr2
strain (see
RESULTS). This 1.4-kb PCR product was digested with XhoI and
SacI and ligated into vector pAD4 M that had been digested
with SalI and SacI, yielding pADH-EXG1
(pAM88). To express PKC1 from its own promoter on a high
copy number (2-µm DNA) plasmid, a 4.2-kb NsiI fragment
containing the entire open reading frame of PKC1 as well as
560 base pairs upstream of the ATG and 175 base pairs downstream of the
stop codon, was cut out of a genomic clone obtained by F. Owen Fields
(this laboratory; Fields, 1991
; Fields and Thorner, 1991
) and ligated
into the URA3-based plasmid YEp352 that had been linearized
with PstI, yielding plasmid pPKC-PN, or into the
LEU2-based plasmid YEp351 that had been linearized with
PstI, yielding plasmid pFR32. To express BCK1-20
in a LEU2-based plasmid, the PvuI-PvuI
fragment of plasmid pRS314-BCK1-20 (Lee and Levin, 1992
)
was replaced by the equivalent LEU2-containing fragment of
plasmid pRS315, yielding plasmid pRS315-BCK1-20.
Selection and Analysis of Chromosomal Suppressor Mutations
A genomic DNA library containing
Tn3::LacZ::LEU2 insertions (generous
gift of Michael Snyder, Department of Biology, Yale University)
was introduced into strain YPT40 (ypk1-1ts
ykr2
) (Casamayor et al., 1999
) by selecting
transformants on SCGlc-Leu medium at 26°C. Temperature-resistant
clones were then selected by their ability to grow at 35°C. Plasmids
carrying genomic DNA corresponding to the sites of insertion were
recovered as described in detail previously (Ross-Macdonald et
al., 1999
) and characterized by direct nucleotide sequence analysis.
| |
RESULTS |
|---|
|
|
|---|
Ypk1 Has a More Prominent Role in Maintaining Viability Than Ykr2
The two protein kinases encoded by the YPK1 and
YKR2/YPK2 genes are very similar to each other and
functionally redundant at the genetic level (Chen et al.,
1993
; Casamayor et al., 1999
). Yeast cells missing either
Ypk1 or Ykr2 are viable, but cells lacking both proteins are inviable,
indicating that these enzymes share an essential function. However,
several observations suggest that Ypk1 plays the predominant role in
executing this essential function. First, ypk1
mutants
are slow growing at 30°C (Maurer, 1988
; Chen et al.,
1993
), whereas ykr2
cells do not display any obvious
growth phenotype, compared with otherwise isogenic
YPK1+ YKR2+
control cells (Figure 1). Moreover, we
found that the slow-growth phenotype of ypk1
cells is
strongly exacerbated at lower temperatures, even at 26°C (Figure 1).
In essence, ypk1
mutants are cold sensitive and
ykr2
mutants are not.
|
Among the close mammalian relatives of Ypk1 and Ykr2 is p70 S6 kinase
(50% identity in the catalytic domain). Activation of mammalian p70 S6
kinase by mitogens is blocked by rapamycin, an immunosuppressive drug
(Chung et al., 1992
; Kuo et al., 1992
; Thomas,
1993
). Therefore, we tested whether loss of either Ypk1 or Ykr2
conferred on yeast cells elevated sensitivity to this agent. Compared
with wild-type cells, we found that ypk1
cells, but not
ykr2
cells, were hypersensitive (~10-fold) to the
growth inhibitory effect of rapamycin (Schnieders, 1996
; Torrance,
2000
), providing a second distinction between ypk1
and
ykr2
mutants. We found that ypk1
cells (but
not ykr2
cells) were also hypersensitive to hygromycin B,
valinomycin, polyoxin D, cycloheximide (Torrance, 2000
), and caffeine
(Bezman, unpublished observations). These results suggested that
ypk1
cells are generally more permeable to drugs and
provided indirect evidence for a defect or perturbation in the cell
envelope in ypk1
cells (see below). When overexpressed, either YPK1 or YKR2 was able to restore the
normal level of drug sensitivity (Roelants, unpublished observations).
Given the fact that Pkh1 and Pkh2 are upstream activators of Ypk1 and
Ykr2 (Casamayor et al., 1999
), it was of interest to test
whether loss of either Pkh1 or Pkh2 might also confer a drug-sensitive
phenotype. However, neither a pkh1
mutant nor a
pkh2
mutant showed any degree of hypersensitivity to the
compounds mentioned above, compared with the parental strain (Torrance,
2000
).
Catalytically inactive (kinase-dead) alleles of protein kinases
frequently act as dominant-negatives (Herskowitz, 1987
). Hence, we
tested whether overexpression of catalytically inactive versions of
Ypk1 and Ykr2 would be toxic to cells. We constructed two such derivatives altered in the invariant Lys found in conserved kinase motif II (Hanks and Hunter, 1995
). We showed that Ypk1(K376A) and
Ykr2(K373A) are indeed catalytically nonfunctional in vitro and unable
to complement the lethality of ypk1
ykr2
cells in vivo
(Torrance, 2000
). To test whether these alleles behave in a
dominant-negative manner when overexpressed from an inducible promoter,
plasmids pGAL-YPK1-(K376A-KD) and
pGAL-YKR2-(K373A-KD) were introduced into
wild-type cells (YPH499) and into ypk1
(YES3) and
ykr2
(YES1) mutants, selecting for transformants on
SC-Leu medium containing Glc as the carbon source. The resulting
transformants then were streaked onto SC-Leu medium containing Gal/Suc
to select for maintenance of the plasmid and to induce expression of
either kinase-dead Ypk1 or kinase-dead Ykr2. High-level expression of catalytically inactive Ypk1 was growth inhibitory to all three cell
types; however, the strongest growth inhibition was observed in
ypk1
cells and the mildest was observed in wild-type
cells (Figure 2A). In contrast,
high-level expression of catalytically inactive Ykr2 had no detectably
detrimental effect on growth in any of the strains (Figure 2A). To rule
out the possibility that this differential effect was due to a
difference in the level of expression of these proteins, Ypk1,
Ypk1(K376A), Ykr2, and Ykr2(K373A) were tagged at their C termini with
a c-Myc epitope and introduced into ypk1
or
ykr2
strains. Identical amounts of protein from extracts
of the resulting transformants were immunoprecipitated with anti-Myc
mAb 9E10 antibodies, resolved by SDS-PAGE, and visualized by
immunoblotting with polyclonal anti-Ypk1 or anti-Ykr2
antibodies. This analysis verified that the proteins were expressed at
equivalent levels (Figure 2B). The fact that kinase-dead Ypk1 was able
to effectively impede its own function and that of Ypk2, and the fact
the converse was not true, provided a third independent indication that
Ypk1 plays the more predominant role.
|
Pkh1 Preferentially Activates Ypk1 and Pkh2 Preferentially Activates Ykr2
Purified Pkh1 phosphorylates and activates purified Ypk1 in vitro
(Casamayor et al., 1999
). To examine the state of activation of Ypk1 and Ykr2 in cell extracts and its dependence on the function of
Pkh1 and Pkh2, we developed an immune-complex kinase assay. Cell
extracts were prepared from strains expressing c-Myc epitope-tagged derivatives of Ypk1 or Ykr2, immunoprecipitated with anti-Myc mAb 9E10,
and samples of the resulting immunoprecipitates were examined for
protein content by SDS-PAGE and for catalytic activity using a specific
peptide substrate (Cross-tide) and [
-32P]ATP
in a filter binding assay (Alessi et al., 1995
). As
independent negative controls to assess the nonspecific background,
extracts were prepared from cells expressing untagged Ypk1 and Ykr2 and from cells expressing kinase-dead derivatives of Ypk1 and Ykr2. Immune
complexes from wild-type cells expressing wild-type Ypk1-myc showed
~10-fold increase in phosphotransferase activity compared with both
negative controls: immune complexes from wild-type cells expressing
untagged Ypk1 (Figure 3A, top) and immune
complexes from wild-type cells expressing catalytically inactive
Ypk1-myc (Torrance, 2000
). When Ypk1-myc was isolated from
pkh1
cells, however, the increase in activity was
reproducibly 40-60% lower than that observed in wild-type cells,
whereas within experimental error, the recovery of Ypk1-myc activity
was unaffected in pkh2
cells (Figure 3A, top). Most
tellingly, activity was greatly increased (
4-fold) when Ypk1-myc was
isolated from cells cooverexpressing Pkh1 but only modestly elevated
(~1.5-fold) when Pkh2 was cooverexpressed (Figure 3A, top). Because
Ypk1-myc and Pkh1 (or Pkh2) were both expressed from multicopy plasmids
carrying the GAL1 promoter, which compete for a limiting
pool of the Gal4 transactivator, the total amount of Ypk1 produced was
reduced (Figure 3A, bottom); thus, the increase in specific activity
when Ypk1-myc and Pkh1 were co-overexpressed is even more dramatic than
the observed increase in total activity. Moreover, immunoprecipitated
Ypk1-myc ran as a set of multiple bands that were collapsed into a
single band of faster mobility upon treatment with a phosphatase
(Figure 3A, bottom). Phosphatase-treated samples were no longer
catalytically active (Torrance, unpublished observations). These
findings suggest that activation is due to phosphorylation and that
activation of Ypk1 is more dependent on Pkh1 than on Pkh2.
|
Immune complexes from wild-type cells expressing wild-type Ykr2-myc
showed approximately a 10-fold increase in phosphotransferase activity
compared with both negative controls: immune complexes from wild-type
cells expressing untagged Ykr2 (Figure 3B) and immune complexes from
wild-type cells expressing catalytically inactive Ykr2-myc (Torrance,
2000
). When Ykr2-myc was isolated from either pkh1
or
pkh2
cells, however, the increase in activity was
reproducibly lower than that observed in wild-type cells, suggesting
that both Pkh1 and Pkh2 contribute to phosphorylation and activation of
Ykr2. Total activity was stimulated approximately threefold when
Ykr2-myc was recovered from cells co-overexpressing Pkh2, but only
~1.5-fold when Pkh1 was co-overexpressed (Figure 2B), indicating that
phosphorylation and activation of Ykr2 are more responsive to Pkh2 than
to Pkh1.
Loss of PKH2 (but Not PKH1) Exacerbates Slow Growth of ypk1
Cells
The biochemical assays discussed above indicated that Pkh1
preferentially activates Ypk1 and Pkh2 preferentially activates Ykr2.
Given that both phk1
pkh2
and ypk1
ykr2
double mutants are inviable (Casamayor et al.,
1999
), if the discrimination observed in vitro with overexpressed
proteins is even more stringent in vivo when these enzymes are
expressed at their normal levels then it might be expected that certain
mutant combinations might display genetic interaction. To construct all
possible double mutant combinations between either ypk1
or ykr2
and either pkh1
or
pkh2
, a ypk1
haploid was crossed to a
pkh1
strain and to a pkh2
strain to create
two diploid strains: PKH1/pkh1
::TRP1
YPK1/ypk1
::HIS3 and
PKH2/pkh2
::HIS3 YPK1/ypk1
::TRP1.
Likewise, a ykr2
haploid was crossed to a
pkh1
strain and to a pkh2
strain to create two additional diploid strains: PKH1/pkh1
::TRP1
YKR2/ykr2
::HIS3 and
PKH2/pkh2
::HIS3 YKR2/ykr2
::TRP1.
The four doubly heterozygous diploid strains were sporulated. After
tetrad dissection, viable Trp+
His+ haploid spores were readily recovered from
all four diploids, indicating that all four double mutants
(pkh1
ypk1
, pkh2
ypk1
, pkh1
ykr2
, and pkh2
ykr2
) are viable (Figure
4A). However, all of the pkh2
ypk1
spore clones grew significantly more slowly than any of
the ypk1
spore clones or any of the pkh2
spore clones, when either streaked to single colonies on plates (Figure
4A) or examined by more definitive spot tests (Figure 4B). This finding provides genetic evidence in support of the biochemical results that
the primary and physiologically relevant upstream activator of Ykr2 in
vivo is Pkh2 (and not Pkh1). Nonetheless, a yeast cell can survive with
either member of these two tiers of protein kinases. Hence, Pkh1 and
Pkh2 must each be able to phosphorylate and activate either Ypk1 or
Ykr2 to at least some significant degree.
|
Loss of PKH1 (but Not PKH2) Alleviates Toxicity of Hyperactive Ypk1
Ypk1 and Ykr2 share 88% identity within their 252-residue kinase
domains and 75% identity within their downstream 75-residue C-terminal
extensions. Ypk1 and Ykr2 also share considerable similarity within
their 350-353-residue amino-terminal extensions: 22% identity within
the first ~100 residues and strikingly, 65% identity within the next
250 residues. To investigate what the large amino-terminal domain might
contribute to the function of Ypk1, a truncation allele of Ypk1 was
constructed in which the entire amino terminus (residues 2-336) was
deleted. The YPK1(
2--336) allele, encoding Ypk1-
N,
commences with Ser337; in Ypk1, the first Gly of the GxGxxG motif
conserved in all protein kinases (Hanks and Hunter, 1995
) lies at
residue 354. To permit its conditional expression, YPK1(
2-336) was inserted in a multicopy vector under
control of the GAL1 promoter. Induction of the resulting
plasmid, pGAL-YPK1-
N (pAM101), in a wild-type
strain (W303-1B) on galactose-containing medium was toxic as judged by
the exceedingly slow growth of single colonies (Figure
5). Despite its toxicity, overexpression
of Ypk1-
N was capable of restoring growth (albeit very slowly) to an
otherwise inviable ypk1
ykr2
double mutant (Torrance,
2000
). The observed toxicity required the catalytic activity of
Ypk1-
N because a kinase-dead derivative, Ypk1(K376A)-
N, was not
detectably growth inhibitory (Figure 4), even though
immunoblotting indicated that, after induction,
Ypk1-
N and Ypk1(K376A)-
N were expressed at equivalently high
levels (Torrance, unpublished observations). Revealingly, the toxicity
of Ypk1-
N was also alleviated in cells lacking Pkh1, but not in
cells lacking Pkh2 (Figure 5). The fact that Pkh1 was required for the
dominant toxicity of Ypk1-
N provides genetic evidence in support of
the biochemical results that the primary and physiologically relevant
upstream activator of Ypk1 in vivo is Pkh1 (and not Pkh2).
|
Differential Subcellular Localization of Pkh1, Pkh2, Ypk1, and Ykr2
One explanation for the observed preferential phosphorylation of
Ypk1 by Pkh1, and of Ykr2 by Pkh2, is that the activating enzyme and
its downstream target are confined to the same subcellular compartment.
As an initial approach to examine localization, each of these four
proteins was tagged at its N terminus with three tandem in-frame
repeats of GFP and expressed from the GAL1 promoter in a
multicopy plasmid. Both 3GFP-Pkh1 and 3GFP-Pkh2 were able to complement
the temperature sensitivity of a pkh1ts
pkh2
strain at restrictive temperature (37°C) on
galactose-containing medium and even on glucose-containing medium
(Roelants, unpublished observations), indicating that each construct
was functional. Similarly, 3GFP-Ypk1 and 3GFP-Ykr2 retained their
biological function (and transcriptional control was tighter) because
each construct was able to complement the temperature sensitivity of
ypk1-1ts ykr2
cells at the nonpermissive temperature
(37°C) on galactose-containing medium (but not on glucose-containing
medium) (Roelants, unpublished observations). In addition, as judged by
immunoblotting with anti-GFP antibodies, each of the
four tagged proteins was expressed intact and had the molecular weight
expected for the full-length chimeric protein (Roelants, unpublished observations).
Live wild-type cells expressing each of the four fusions to 3GFP were
examined under the fluorescence microscope (Figure
6). The 3GFP-Ypk1 chimera was found
exclusively in the cytosol and was excluded from both the vacuole
(whose position was observed by phase contrast microscopy of the same
field) and the nucleus (whose position was revealed by growing the
cells in the DNA-specific dye DAPI). In contrast, the 3GFP-Ykr2 chimera
accumulated in the nucleus, congruent with the DAPI-stained DNA,
although it was also readily detectable in the cytoplasm.
Interestingly, when fused to 3GFP, the catalytic domain of Ypk1
(Ypk1
N), which by itself is toxic when overexpressed (see above),
was located predominantly in the nucleus, unlike full-length 3GFP-Ypk1,
suggesting that its toxicity may arise largely from its
mislocalization. The same patterns of distribution for Ypk1 and Ykr2
were also observed if the cells were fixed, permeabilized, and stained,
respectively, with polyclonal anti-Ypk1 and anti-Ykr2 antibodies
(Torrance, unpublished observations). Likewise, identical patterns of
distribution were observed when cells expressing Ypk1-myc or Ykr2-myc
were examined by indirect immunofluorescence using anti-c-Myc mAb 9E10 (Roelants, unpublished observations).
|
As observed for 3GFP-Ypk1, 3GFP-Pkh1 was localized exclusively to the cytosol, and clearly excluded from both the vacuole and the nucleus (Figure 6). The most prominent feature of the 3GFP-Pkh1 staining was, however, bright puncta or larger patches situated at the cell cortex. These dots are not congruent with actin patches, as was revealed by costaining with rhodamine-labeled phalloidin (Roelants, unpublished observations). The same distribution pattern was observed if cells expressing Pkh1-(HA)3 were fixed, permeabilized, and examined by indirect immunofluorescence by using an anti-HA mAb and an appropriate fluorescently tagged secondary antibody (Roelants, unpublished observations). Unlike 3GFP-Pkh1, 3GFP-Pkh2 was not excluded from the nucleus, but like 3GFP-Pkh1, the most prominent feature of the staining was a large number of punctate bodies immediately subtending the plasma membrane (Figure 6), which were also distinct from actin patches (Roelants, unpublished observations).
Thus, taken together, these observations indicate that Pkh2 and Ykr2 are able to enter a compartment (the nucleus) from which Pkh1 and Ypk1 are normally excluded. Thus, these findings help to explain, at least in part, the greater dependence of Ypk1 activation on Pkh1 and the greater dependence of Ykr2 activation on Pkh2.
Genetic Analysis of Ypk1 and Ykr2 Function by Selection of Dosage Suppressors
To identify gene products that may be involved in processes both
upstream and downstream of Ypk1 and Ykr2, we selected, first, for genes
that when overexpressed from a URA3-marked multicopy vector,
were able to restore growth to ypk1-1ts
ykr2
cells at an otherwise nonpermissive temperature
(35°C), as described in MATERIALS AND METHODS. From 20,000 Ura+ transformants, we recovered 18 plasmids that
were able to support growth reproducibly at the restrictive temperature
(Table 2). As expected, seven independent
isolates of YKR2 and one isolate of YPK1 were
obtained. Two of the other suppressor genes obtained, one encoding a
putative chaperone (HLJ1) and the other encoding a component
of RNA polymerase II holoenzyme (SRB4), may rescue because
they stabilize or elevate expression of the temperature-sensitive Ypk1-1 enzyme, although this hypothesis was not tested directly. Another suppressor plasmid carried the YPC1 gene, which
encodes an enzyme that can generate phytosphingosine from the
corresponding phytoceramide (Mao et al., 2000
) and hence
presumably rescues by hyperstimulating Pkh1 and Pkh2. Indeed, we have
shown previously that elevated Pkh1 can restore growth to
ypk1-1ts ykr2
cells at
otherwise restrictive temperature (Casamayor et al., 1999
).
Indeed, when excised from the original isolate and overexpressed from a
completely different vector, YPC1 rescues the temperature
sensitivity of ypk1-1ts ykr2
cells (Roelants, unpublished observations). In contrast, at least one
other of the genes from the same insert, RPS6B, was not a
suppressor on its own (Roelants, unpublished observations).
|
Revealingly, among the seven remaining dosage suppressors, two plasmids
carried a single intact open reading frame corresponding to the
EXG1 gene, which encodes the major exo-
(1,3)-glucanase involved in cell wall remodeling (Larriba et al., 1995
).
Indeed, when excised from the original isolate and expressed from a
constitutive promoter (ADH1) in a completely different
multicopy vector (see MATERIALS AND METHODS), elevated expression of
EXG1 reproducibly suppressed, albeit weakly, the
temperature-sensitive growth defect of ypk1-1ts
ykr2
cells, even at 37°C (Figure
7A). Also obtained were two isolates of a
locus (YBL104c) of unknown function, but which seems from the phenotype
of a null allele to also have effects on cell wall structure (Obermaier
et al., 1995
). Another suppressor plasmid isolated carries
multiple open reading frames, one of which is a candidate chitinase
(Jacq et al., 1997
), which may also influence cell wall
structure. At least one of the other genes carried on this same
plasmid, FRQ1 (Hendricks et al., 1999
), is not
responsible for the suppression and does not contribute to the
suppression (Roelants, unpublished observations). The final two dosage
suppressors encoded proteins that might act by enhancing the efficiency
with which enzymes involved in cell wall biosynthesis or remodeling are
delivered to their final destination and/or are activated there. One
plasmid carried GOT1, which specifies a membrane protein thought to enhance the function of a t-SNARE heavy chain, Sed5, involved in vesicle-mediated protein transport from the endoplasmic reticulum (ER) to the Golgi (Conchon et al., 1999
). The
other plasmid carried only PLB1, which specifies the
phospholipase B that is primarily responsible for the conversion of
phosphatidylcholine and phosphatidylethanolamine in the exocellular
leaflet of the plasma membrane to lysophosphatidylcholine (and
glycerophosphocholine) and lyso-phosphatidylethanolamine (and
glycerophosphoethanolamine), respectively (Lee et al.,
1994
). It is well documented that the activity of many classes of
membrane-associated enzymes can be influenced dramatically (either
stimulated or inhibited), depending on the nature of the phospholipids
(or their derivatives) with which those enzymes associate (Dowhan,
1997
).
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