The Inactive Form of a Yeast Casein Kinase I Suppresses the
Secretory Defect of the sec12 Mutant
IMPLICATION OF NEGATIVE REGULATION BY THE Hrr25 KINASE IN THE
VESICLE BUDDING FROM THE ENDOPLASMIC RETICULUM*
Akiko
Murakami
§,
Keitarou
Kimura¶, and
Akihiko
Nakano
From the
Molecular Membrane Biology Laboratory,
RIKEN, Wako, Saitama 351-0198 and the ¶ Genetic Engineering
Laboratory, National Food Research Institute, Tsukuba,
Ibaraki 305-8642, Japan
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ABSTRACT |
Sec12p is the guanine nucleotide exchange factor
of Sar1 GTPase and functions at the very upstream in the vesicle
budding reactions from the endoplasmic reticulum (ER). We previously
identified three yeast loci, RST1, RST2, and
RST3, whose mutations suppressed the temperature-sensitive
growth of the sec12-4 mutant (Nakano, A. (1996)
J. Biochem. (Tokyo) 120, 642-646). In the
present study, we cloned the wild-type RST2 gene by
complementation of the cold-sensitive phenotype of the
rst2-1 mutant. RST2 turned out to be identical to HRR25, a gene encoding a dual-specificity casein kinase
I in yeast. The rst2-1 mutation, which is now renamed
hrr25-2, was due to the T176I amino acid replacement in
the kinase domain. This mutation remedied not only the
temperature-sensitive growth but also the defect of ER-to-Golgi protein
transport of sec12. Immunoprecipitation of the
hemagglutinin-tagged Hrr25-2 protein and a subsequent protein kinase
assay showed that the kinase activity of the mutant protein was
markedly reduced. The overproduction of another kinase-minus mutant of
Hrr25p (Hrr25p K38A) slightly suppressed the growth defect of
sec12-4 as well. These observations suggest that the
reduction of the kinase activity in the mutant protein is important for
the suppression of sec12. We propose that Hrr25p negatively
regulates the vesicle budding from the ER.
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INTRODUCTION |
The secretory pathway begins from the
ER.1 By genetic approaches
with yeast Saccharomyces cerevisiae, many secretory genes have been identified in the whole pathway, and more than 20 genes are
now known to function in the transport from the ER to the Golgi
apparatus. Among these genes, SEC12 and SAR1 are
believed to play pivotal roles in the earliest step, that is the
formation of transport vesicles from the ER. SAR1 encodes a
21-kDa GTPase (Sar1p) (1), which functions as a molecular switch to
recruit a coat protein complex, COPII, onto the ER membrane (2);
SEC12 codes for a 70-kDa integral membrane protein (Sec12p)
in the ER and acts as the guanine nucleotide exchange factor (GEF)
toward Sar1p, which converts Sar1p from the inactive GDP form to the active GTP form (3). Sar1p-GTP promotes the assembly of COPII (Sar1p,
Sec13p/Sec31p, and Sec23p/Sec24p) (2), budding, formation, and release
of vesicles (2, 4). Thus, Sec12p is the most upstream player in the
vesicle budding from the ER as far as we know. However, little is known
as to when and how the GEF activity of Sec12p is triggered in this
earliest event of vesicle budding. The regulation of Sec12p function
may be a key issue for understanding the mechanisms of cargo and
resident selection in the vesicle budding event.
We recently identified three genetic loci, RST1,
RST2, and RST3, whose mutations suppressed the
temperature-sensitive (ts) growth defect of the sec12-4
mutant (5). These genes were expected to be the candidates of Sec12p
regulators. RST1-1 was a dominant mutation and caused
elevated expression of Sec12p. rst2 and rst3 were
recessive and gave pleiotropic phenotypes including slow growth at low
temperature, aggregation of cells, and heterogeneous glycosylation of
Sec12p. In this study, we extended characterization of the
rst2-1 mutant and cloned the wild-type RST2 gene
by complementation.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Culture Conditions--
The yeast strains used
in this study were MBY10-7A (sec12-4 ura3-52 leu2-3, 112 trp1-289 his3 his4 suc gal2 MATa) (6), MBY10-7C
(sec12-4 ura3-52 leu2-3, 112 trp1-289 his3 his4 suc gal2 MAT
) (6), ANY21 (ura3-52 leu2-3, 112 trp1-289 his3
his4 suc gal2 MATa) (6), STR2 (sec12-4 rst2-1
ura3-52 leu2-3, 112 trp1-289 his3 his4 suc gal2
MATa) (5), and AMY7-4B (2µ HRR25 URA3;
hrr25::HIS3 ura3-52 lys2-801 ade2-101 trp1-D63 his3-D200 leu2-D1 MATa) (this study). The cells were
grown at 37 °C (for STR2), 30 °C or 23 °C (for MBY10-7A and
MBY10-7C) in YP medium (2% (w/v) polypeptone (Nihon Pharmaceutical
Co. Ltd., Tokyo, Japan), 1% (w/v) yeast extract (Difco Laboratories,
Inc., Detroit, MI)) containing 2% (w/v) glucose (YPD) or in MC medium (0.67% (w/v) yeast nitrogen base without amino acids (Difco
Laboratories), and 0.5% (w/v) casamino acids (Difco Laboratories))
containing 2% (w/v) glucose (MCD) supplemented appropriately. For
derepression of the GAL1 promoter, MC medium was
supplemented with 5% galactose and 0.2% sucrose (MCGS).
Plasmids--
A yeast replication plasmid (pJJ215), a
single-copy plasmid (pRS314), a multicopy plasmid (pYO324), and an
integration plasmid (pRS304) were described previously (7-9). Another
multicopy plasmid, pQR324, was provided by H. Qadota of the Nara
Institute of Science and Technology.
Cloning of the RST2 Gene--
For the cloning of the
RST2 gene, the strain STR2 (rst2-1 sec12-4),
which shows a Cs
Ts+ phenotype, was
transformed with a genomic library (10) constructed on YEp13, which
contains the LEU2 gene as the selectable marker. Electroporation was used to obtain high transformation efficiency (see
below). Transformants were plated in 5-6 ml of regeneration top agar
containing 1× MVD, 1 M sorbitol, 2.5% agar, and
appropriate supplements and incubated at 24 °C. After 5-6 days,
large colonies were picked up, streaked, and further incubated at 15 and 37 °C. Among eight candidates we obtained, one clone (named
P2-3) reproducibly conferred Cs+Ts
growth to
the STR2 cells. This plasmid was recovered from the yeast transformant.
After the confirmation of the phenotypes by retransformation, the
genomic insert was subcloned and subjected to further complementation tests.
Transformation by Electroporation--
From a saturation culture
in 50 ml of YPD, STR2 cells were harvested and washed two times with
sterile distilled water. The cells were resuspended in 20 ml of 0.1 M Tris-HCl, pH 9.4, and 100 µl of 1 M DTT.
After incubation at 30 °C for 10 min, the cells were centrifuged at
1800 × g for 5 min and resuspended in the spheroplasting buffer (0.67% yeast nitrogen base, 1 M
sorbitol, and 0.5% glucose). Zymolyase-100T (Seikagaku Corp.) (5 mg)
was added to the cell suspension, and incubation was continued for 10-20 min at 30 °C. The Zymolyase-treated samples were overlaid on
25 ml of 1.4 M sorbitol solution and centrifuged at
1800 × g. Spheroplasted pellets were resuspended in
0.5 ml of 1 M sorbitol and kept on ice until use for electroporation.
For electroporation, 4 µg of DNA (up to 10 µl) was added to a
100-µl aliquot of the spheroplast suspension. The samples were mixed
gently and put on ice 5 min prior to electroporation, and then
transferred to a cold sterile cuvette (0.4 cm) and subjected to
electroporation (Bio-Rad Gene PulserTM; pulse at 1.5 kV, 25 microfarads, 200 watts for 3 s). Immediately after
electroporation, 100 µl of 1 M sorbitol solution was
added and the samples were spread on MVD plates (with adequate
supplements) containing 1 M sorbitol.
Introduction of the Influenza Hemagglutinin (HA) Tag into
HRR25--
The AflII-AflII fragment of
HRR25 was amplified with an NheI site created
near the 5'-terminus of the ORF by PCR (primer sequences: 5'-TCTATGGACTTAAGAGCTAGCGTAGGAAGGAAATTT-3' and
5'-TCTACCGCTTAAGTATCTGTAGACGCG-3'). The resulting NheI
site-containing fragment was used to replace the original HRR25
AflII-AflII fragment. The DNA cassette encoding three
tandem repeats of the HA epitope was excised from pYT11 by
NheI digestion (11), and inserted into the NheI
site of the above construction. The resulting plasmid was named pAM5-2
(2µ 3HA-HRR25 TRP1). The expression of
3HA-HRR25 was confirmed by immunoblotting with the
monoclonal anti-HA antibody 16B12 (Berkeley Antibody). A plasmid
harboring 3HA-rst2-1, pAM6-2 (2µ 3HA-rst2-1 TRP1), was constructed from pAM1-5 (2µ rst2-1 TRP1)
by substituting the AflII-AflII fragment of
pAM5-2 for that of pAM1-5. pAM1-5 is the plasmid containing the
mutant allele rst2-1, which was obtained by the allele
recovery method.
The HRR25 expression plasmid, pAM4-1 (2µ GAL1
promoter 3HA-HRR25 URA3), was constructed as follows. A
HindIII site was created just before the initiation codon of
HRR25 by PCR. The BamHI-HindIII fragment containing 3HA-HRR25 was subcloned into pYES2
(Invitrogen, Leek, The Netherlands), which is a 2µ-based multicopy
plasmid carrying the GAL1 promoter and a selectable
URA3 marker.
Site-directed Mutagenesis in 3HA-HRR25--
The K38A mutation in
3HA-HRR25 was created by PCR with the following primers: for
N-terminal fragment, 5'-GGCTCGAGGAAAGCATTTTGG-3' (hrr25 XHOI) and
5'-CGATTCCAGAGCGATGGCTAC-3' (hrr25 XHOI KA); for C-terminal fragment,
5'-GTAGCCATCGCTCTGGAATCG-3' (hrr25 NCOI KA) and
5'-CTCGGATCCCCATGGCAAAGAACCCTT-3' (hrr25 NCOI). The mutation was
introduced by the first PCR with hrr25 XHOI KA/hrr25 XHOI and hrr25
NCOI KA/hrr25 NCOI and the produced fragments were amplified by the
second PCR with hrr25 XHOI and hrr25 NCOI. The resulting 0.9-kb
XhoI-NcoI fragment was used to replace the
corresponding region of 3HA-HRR25 to yield the
3HA-HRR25 K38A mutant allele.
Immunoprecipitation of HA-Hrr25p and Its Kinase
Assay--
Immunoprecipitation kinase assays with HA-tagged Hrr25p
were performed by the method described (12, 13) with some
modifications. The HRR25 deletion strain (AMY7-4B) was
transformed with plasmids containing 3HA-HRR25, 3HA-rst2-1,
or non-tagged HRR25 (2µ TRP1) and grown in YPD.
Cells in a 100-ml culture were harvested at 2 OD600/ml and
resuspended in 500 µl of IPK buffer (50 mM Tris-HCl, pH
7.5, 1% Nonidet P-40, 0.05% SDS, 0.05% sodium deoxycholate, 5 mM EDTA, 5 mM DTT, 100 mM NaCl, 5 µg/ml each of leupeptin, antipain, chymostatin, and pepstatin, and
phosphatase inhibitor (0.2 mM sodium orthovanadate, 30 mM sodium pyrophosphate, and 50 mM sodium fluoride)). Glass beads (200 mg) were added to the cell suspension in
2-ml screw-capped tubes, and the samples were lysed by vigorous vortexing without heating. The materials were centrifuged and supernatants were transferred to fresh screw-capped tubes containing 300 µl of IPK buffer. After the addition of 2 µl of 1 mg/ml anti-HA monoclonal antibody (12CA5; Boehringer Mannheim) to each 200 µg of
total protein, the samples were kept on ice for 2 h and mixed with
10 µl of 50% Protein A-Sepharose CL-4B at 4 °C for 2 h.
Immune complexes were collected by centrifugation for 10 s in a
microtube, and washed twice with IPK buffer, twice with 1 M
NaCl in IPK buffer without inhibitor, and twice with kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2,
and 1 mM DTT). Each sample was resuspended in 15 µl of
kinase buffer and split 1:2; one third was transferred to a fresh
screw-capped tube for kinase assays, and the remainder was spun down,
aspirated dry, boiled briefly in the 1× SDS-PAGE sampling buffer, and
subjected to SDS-PAGE and immunoblotting with the anti-HA antibody.
The immunoprecipitates for the kinase assay were washed again with
kinase buffer, dried, and resuspended in 15 µl of kinase buffer prior
to preincubation at 37 °C for 10 min. The kinase assay was initiated
by adding 5 µl of kinase buffer containing 10 mCi of
[
-32P]ATP (final ATP concentration = 1 mM; 3000 Ci/mmol; NEN Life Science Products). Reactions
were terminated after 15 min with 2 µl of 4× SDS-PAGE sampling
buffer and heated to 95 °C for 2 min before electrophoresis. The
gels were dried and visualized by autoradiography using BAS2500 (Fuji Film).
 |
RESULTS |
rst2 Suppresses the Secretory Defect of the sec12 Mutation--
To
examine whether rst2-1 suppresses not only the
temperature-sensitive growth defect but also the secretory defect of
the sec12-4 mutation, we performed a pulse-chase and
immunoprecipitation experiment with the anti-carboxypeptidase Y (CPY)
antibody (Fig. 1, upper
panel). In the wild-type cells, newly synthesized CPY undergoes stepwise processing from the 67-kDa ER precursor (p1) through
the 69-kDa Golgi precursor (p2) to the 61-kDa mature vacuolar form (m).
When the sec12 mutant cells were pulse-labeled for 4 min and
chased for 2 h at 37 °C, the p1 form accumulated, indicating that the ER-to-Golgi transport was blocked. Such accumulation of the p1
form was not detected when the sec12-4 rst2-1 double mutant cells were incubated at 37 °C. Normal modification and processing from p1 through p2 to m forms was observed, although the
rate was a little slower than that of wild-type cells.

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Fig. 1.
The rst2-1 mutation
restores the secretory defect of sec12 cells.
Cells of MBY10-7A (sec12-4 RST2), ANY21 (SEC12
RST2), and STR2 (sec12-4 rst2-1) were starved for
sulfate for 30 min at the indicated temperatures. After 4-min (for CPY)
or 5-min (for Gas1p) pulse labeling, cells were chased for the
indicated times at the same temperatures. Cell lysates were prepared
and subjected to immunoprecipitation, SDS-PAGE, and fluorography. Times
are shown in minutes, except for the 2-h points. p1, ER
form; p2, Golgi form; m, mature form;
i, immature form.
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We also performed a pulse-chase experiment on a
glycosylphosphatidylinositol-anchored plasma-membrane protein, Gas1p
(14, 15) (Fig. 1, lower panel). Like the case of
CPY, the immature form (i) of Gas1p accumulated in the sec12
mutant cells after 30-min chase at 37 °C. In contrast, Gas1p was
processed to the mature form (m) in the sec12-4 rst2-1
double mutant cells at the restrictive temperature for
sec12, 37 °C. These data indicate that the
rst2 mutation remedies the secretory defect of the
sec12 mutation.
Overproduction of the Sec12 ts protein by the introduction of the
mutant gene on a multicopy plasmid (2µ) allows growth of the
sec12 ts strain at 37 °C (Fig.
2A; see also Ref. 16). Even a
slight increase of the gene dosage by the introduction of the single-copy plasmid (CEN) of sec12 could suppress
the ts growth to some extent. We performed immunoblotting analysis
using the anti-Sec12p antibody to examine whether the level of
Sec12-4p increased in the sec12 rst2 cells to the extent
that was able to suppress the sec12 mutant phenotype. As
shown in Fig. 2B, the amount of Sec12-4p was slightly
increased in the sec12-4 rst2-1 cells as compared with
that in the sec12-4 RST2 cells (lanes
3 and 4 versus lanes
1 and 2). Careful quantification of the results of three independent experiments with the amount of Pgk1p as an internal standard indicated that the amount of Sec12-4p was 2-3 times
larger in sec12-4 rst2-1 than in sec12-4 RST2.
The extent of the increase was almost the same as the case where an
extra copy of sec12-4 was supplied by the introduction of a
single-copy (CEN) plasmid (lanes 5 and
6). The level of Sar1p was not affected either by the
rst2-1 mutation or the introduction of sec12-4
plasmids. These results suggested a possibility that the increase of
Sec12-4p by the rst2-1 mutation was the cause of the
suppression. However, the effects of the rst2-1 mutation
and the increase of Sec12-4 were quite different when the
intracellular transport of CPY was examined. As shown in the
bottom panel of Fig. 2B, the
introduction of the rst2-1 mutation in sec12-4
cells completely remedied the accumulation of the ER form (p1). In
contrast, in the cells in which the Sec12-4 levels were raised by
sec12-4 plasmids (CEN or 2µ), the accumulation
of the p1 CPY was not completely cured even though the ts growth was
almost completely suppressed by 2µ sec12-4. These
observations led us to conclude that the effect of rst2-1
in the suppression of sec12-4 was not due to the increase of the Sec12-4 protein by itself.

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Fig. 2.
Expression of the sec12-4
allele in the sec12-4 and sec12-4
rst2-1 mutant cells. A, the growth of
sec12 ts mutant cells carrying plasmid-borne
sec12-4. Cells of wild-type (ANY21),
sec12-4/2µ sec12-4 (MBY10-7A/pAM13-3),
sec12-4/CEN sec12-4 (MBY10-7A/pAM13-2), and
sec12-4/vector (MBY10-7A/pYO326) were incubated at the
indicated temperatures for 4 days. B, immunoblotting of
sec12 ts mutant cells carrying plasmid-borne single-copy
(CEN) or multicopy (2µ) sec12-4 with
antibodies against Sec12p, Sar1p, Pgk1p, and CPY. For the case of
Sec12p, the lysates were treated with endoglycosidase H to remove
N-linked oligosaccharides. Cells of sec12-4 RST2
(MBY10-7A), sec12-4 rst2-1 (STR2),
sec12-4/CEN sec12-4 (MBY10-7A/pAM13-2), and
sec12-4/2µ sec12-4 (MBY10-7A/pAM13-3) were
precultured at 23 °C (for sec12) or 30 °C (for
sec12-4 rst2-1), cultured for 0 and 2 h at 37 °C,
and then harvested and analyzed by immunoblotting. p1 and
m indicate the ER form and the mature form of CPY,
respectively.
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Cloning of the Gene That Complements the rst2 Mutation:
HRR25--
For the cloning of the RST2 gene, STR2, the
original cold-sensitive rst2-1 sec12 mutant strain, was
transformed with a yeast genomic DNA library constructed on the
multicopy plasmid YEp13 (10). DNA clones that rescued the
cold-sensitive growth were selected. One clone named P2-3 showed good
complementation and was analyzed further. The 6.7-kb insert of P2-3
contained two complete ORFs, HRR25 and TPK2/PKA3
(see Fig. 3A). To localize the
complementation activity of rst2-1 in this insert, deletion analysis was performed. Various fragments from P2-3 were subcloned into a multicopy plasmid, pQR324, or a single-copy CEN
plasmid, pRS314, and introduced into the rst2-1 sec12-4
mutant (STR2). Transformants were tested for growth at the restrictive
temperature for rst2-1, 15 °C. As shown in Fig.
3A, DNA fragments always complemented the rst2
mutant when they contained HRR25. The presence or absence of
TPK2 did not correlate with the complementation activity.
The same results were obtained for both the multicopy and single-copy plasmids. Thus, it is the HRR25 gene that complemented
rst2-1. STR2 cells show morphological abnormality as well
(5). The cells do not separate very well after division and tend to
aggregate either in liquid or on plate culture. The DNA fragment
containing HRR25 not only resumed the growth defect of
STR2 at 15 and 23 °C (Fig. 3B), it also
remedied this aggregation phenotype (Fig. 3C).

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Fig. 3.
Cloning of RST2 and its
identification as HRR25. A, subclones
of the original clone, P2-3, were examined for complementation of the
growth defect of rst2-1. The thick
arrow indicates the coding region of HRR25 and
its direction of transcription. The dashed regions are
derived from the vector, YEp13. Bars below show subclones.
Note that HRR25 is necessary and sufficient for the ability
to complement rst2-1. Abbreviations of restriction enzymes
are: N, NheI; S, SphI;
Xh, XhoI; Nc, NcoI;
P, PvuII; Hp, HpaI;
H, HindIII; B, BstBI;
RV, EcoRV. B and C, the
cold-sensitive growth defect (B) and the aggregation
phenotype of the rst2-1 cells (STR2) at 30 °C
(C) were completely complemented by HRR25.
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To confirm that HRR25 is the authentic RST2 gene,
the fragment containing HRR25 with the LEU2
marker was integrated at the HRR25 locus in the
sec12-4 mutant, MBY10-7C. The integrant was mated with
STR2 which was transformed by pAM2-326, a URA3-marker multicopy plasmid containing HRR25 derived from pYO326. This
plasmid complemented rst2-1 and dramatically improved the
mating and sporulation efficiency of the mutant. The diploid cells were
sporulated, and the progeny haploid cells were plated on MVD (complete
supplement) containing 5-fluoroorotic acid (FOA) at 27 °C to remove
pAM2-326. Among 160 spores analyzed, 67 spores showed
Leu
Cs
phenotype, 90 spores were
Leu+ Cs+, 1 was Leu+
Cs
, and 2 were Leu
Cs+. This
result indicated that the LEU2 marker was tightly linked to
the Cs+ phenotype, namely HRR25 was linked to
the rst2-1 locus. Therefore, we concluded that
RST2 is identical to HRR25. Finally, we renamed our mutant allele (rst2-1) of HRR25,
hrr25-2.
hrr25-2 (rst2-1) Is a Mutant with a Reduced Kinase
Activity--
HRR25 encodes a dual-specificity casein
kinase I (CKI) (17). We isolated the mutant hrr25-2 gene by
the allele recovery method and determined that it contained a mutation
of the C527T replacement in the nucleotide sequence, which caused T176I
mutation in the amino acid sequence (Fig.
4A). A comparison of the amino acid sequence of Hrr25p with other yeast CKIs is shown in Fig. 4B. The T176I mutation is in the region conserved in all
members of the yeast CKI family. To examine whether hrr25-2
is a kinase-minus (low kinase activity) or constitutively active
mutation, we carried out an in vitro kinase assay for the
Hrr25 protein from the wild-type and mutant cells (Fig.
5). HA-tagged versions of the
HRR25 and hrr25-2 genes were constructed for
this purpose. 3HA-HRR25 complemented the mutant phenotypes
of
hrr25 and hrr25-2 (data not shown). The
3HA-tagged Hrr25p was immunoprecipitated with the anti-HA antibody and
subjected to the kinase assay. The result of SDS-PAGE and
autoradiography is shown in Fig. 5B. By the incubation with [
-32P]ATP, the immunoprecipitated 3HA-Hrr25p
phosphorylated Hrr25p itself and casein as a substrate, but not histone
H1 or myelin basic protein (lanes 2 and
4-6). In clear contrast, virtually no phosphorylated bands
were observed when 3HA-Hrr25-2p was immunoprecipitated (lane 3). The amounts of 3HA-Hrr25p and
3HA-Hrr25-2p in the immunoprecipitates were almost the same as
examined by immunoblotting (Fig. 5A). Thus, we conclude that
hrr25-2 is a mutation with a markedly reduced kinase
activity.

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Fig. 4.
The mutation point of hrr25-2.
A, illustration of the HRR25 gene product
(Hrr25p). T176I is the mutation point of hrr25-2 (=
rst2-1). Note that Hrr25p contains a region rich in proline
and glutamine residues at the C terminus (Pro/Gln-rich). B,
comparison of amino acid sequences in the kinase homology region
between Hrr25p and other yeast CKIs. Black boxes
represent the residues identical among the four CKIs, and the
asterisk indicates the hrr25-2 mutation.
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Fig. 5.
In vitro kinase assay of the Hrr25
T176I and K38A mutant proteins. A, hrr25
cells (AMY7-4B) expressing no-tagged HRR25, 3HA-tagged
HRR25, 3HA-tagged hrr25-2 (T176I), and
3HA-tagged hrr25 K38A were lysed and subjected to
immunoprecipitation with the anti-HA monoclonal antibody (12CA5). The
immunoprecipitates were examined for the amount of HA-tagged Hrr25
protein by immunoblotting with the anti-HA monoclonal antibody (16B12).
B, a kinase assay was performed in the presence of
[ -32P]ATP, and the phosphorylated products were
analyzed by SDS-PAGE and autoradiography. As substrates, casein
(lanes 1, 3, 4, and
7), histone H1 (lane 5), and myelin
basic protein (lane 6) were added to the assay
solution. The mobilities of molecular weight markers are shown on the
left.
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Furthermore, we constructed another mutant version of 3HA-tagged
HRR25 (3HA-hrr25 K38A) by site-directed
mutagenesis, which was also expected to be kinase-minus, and to act
dominantly on the wild-type protein when overproduced. In the
immunoprecipitation kinase assay (Fig. 5, lane
7), in fact, we could not detect any kinase activity of
3HA-Hrr25p K38A. As shown in Fig.
6A, the overproduction of
Hrr25p K38A slightly suppressed the growth defect of
sec12-4 at 35 °C. On the other hand, it inhibited the
growth of sec23-1 and sar1-2 at 30 and
33 °C, respectively, and that of the wild-type slightly at
35 °C.

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Fig. 6.
Overexpression of the dominantly acting
kinase-minus mutant of Hrr25p (3HA-Hrr25p K38A) in the
sec12-4 mutant and hrr25 sec12-4
double mutant cells. A, multicopy (2µ),
GAL1-promoter-driven HA-tagged hrr25 K38A (K38A),
or vector alone (vector) was expressed in sec12-4
(MBY10-7A), sec23-1 (MBY8-20C), sar1-2
(TOY224), and wild-type (ANY21) cells. Overnight liquid cultures of
these transformants were diluted from 1 × 107 to
1 × 103 cells/ml by 10-fold serial dilution
(A, left to right), and 5 µl each of
diluted samples were spotted on MCGS plates. The cells were cultured
for 8 days at the indicated temperatures. B, the
hrr25 sec12-4 mutant cells harboring HRR25
(pAM2-326: URA3-marked multicopy plasmid) were transformed
with multicopy, own-promoter-driven hrr25-2 (T176I),
HA-tagged HRR25 (HRR25), hrr25 K38A
(K38A), or vector alone. The transformants were cultured for 6 days at
26 °C on MCD plates (complete supplements) containing 5-fluoroorotic
acid to remove pAM2-326, and then restreaked on MCD (minus tryptophan)
plates and cultured for 14 days at 26 and 35 °C.
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Reduction of the Kinase Activity but Not Complete Loss of Hrr25p Is
Important for the sec12-4 Suppression--
A HRR25
disruption was generated by inserting the 1.8-kb BamHI
fragment of HIS3 from pJJ215 into the
AflII-NdeI sites of HRR25. This
disrupted gene was introduced on an integration vector into the
wild-type diploid, YPH501. The resulting heterozygous diploid was
sporulated and subjected to tetrad dissection. Fast and slow growing
spores segregated 2:2. All the slow growth spores showed His+ phenotype. The correct integration at the
HRR25 locus was confirmed by Southern blotting (data not
shown). This indicates that these slow growing spores were the
hrr25 mutant. This observation is consistent with the
results of Hoekstra et al. (17), i.e.
HRR25 is not essential but very important for cell
viability.
hrr25 showed extremely slow growth at low
(15 °C) and high (37 °C) temperatures.
If the suppression of sec12-4 by hrr25-2 was
due to the reduction of the Hrr25p kinase activity, the disruption of
HRR25 would also suppress the sec12-4 growth
defect. To test this, the
hrr25 mutant cells harboring
pAM2-326 (2µ HRR25 URA3) were mated with the
sec12-4 mutant (MBY10-7C). This diploid was sporulated,
and tetrads were dissected to obtain segregants of the genotype,
hrr25 sec12-4. The spores showed a 2:2 segregation
pattern regarding the sec12-4 phenotype as marked by growth
Ts
on MCD (
Ura) plates. If these spores were cultured
on FOA-containing plates at 15 °C, they also showed a 2:2
segregation pattern, because
hrr25 cells were extremely
slow in growth at low temperature (15 °C). We selected segregants
that are Ts
on MCD (
Ura) and Cs
on FOA
plates, which have the genotype of
hrr25 sec12-4. The segregants were grown on FOA plates at 27 °C to lose the
HRR25 plasmid and examined for growth at high temperatures.
As shown in Table I, they were all unable
to grow on YPD plates at either 35 or 37 °C. This result indicates
that
hrr25 cannot suppress the sec12 mutation
and suggests that hrr25-2 is not a null mutation in terms
of the sec12 suppression. We also constructed double mutants
of hrr25-2 with sec13-1, sec23-1,
and ret1-1, but no clear suppression was observed.
To further analyze the relationship between the kinase activity and the
sec12 suppression, the two mutant alleles of
HRR25, hrr25 K38A and hrr25 T176I,
were expressed in the
hrr25 sec12-4 double mutant cells.
As shown in Fig. 6B,
hrr25 sec12-4 hardly grew at 35 °C. The mutant cells expressing HRR25 did not
grow at 35 °C, either, due to the sec12-4 mutation.
However, the expression of hrr25 K38A or hrr25
T176I suppressed the growth defect at 35 °C at least to some extent.
 |
DISCUSSION |
In this paper, we have shown evidence for the first time that
Hrr25p, a yeast CKI, is involved in vesicle budding from the endoplasmic reticulum.
Family of Yeast CKI--
CKI is an expanding family of kinases,
which have been proposed to play a variety of roles in many cellular
processes. Among five defined isoforms of mammalian CKI (18-20), for
example, the
isoform has been implicated in the regulation of
secretion (21).
The yeast S. cerevisiae possesses four genes that encode
CKI: YCK1, YCK2, YCK3, and HRR25. The redundant
YCK1 and YCK2 genes are required for cell
viability (22) and morphogenesis (23). Recently, several reports have
suggested that YCK1, YCK2, and YCK3
are involved in vesicular transport through the studies of their
genetic interactions. For example, suppressor mutations of the
yck1
yck2-2 mutant
(yckts) define four subunits of a novel clathrin
AP-like complex, AP-3 (24). The yckts mutant
shows a strong synthetic growth defect with chc1-ts (24), and exhibits ts phosphorylation, ubiquitination, and endocytosis of the
-factor receptor (25). YCK1, YCK2, and
YCK3 suppress the defect of the deletion of GCS1,
a yeast ARF GTPase-activating protein gene (26, 27)
, in cell proliferation from the stationary phase (28).
HRR25 forms an essential gene pair with YCK3 but cannot suppress the
gcs1 mutant. These relationships are
illustrated in Fig. 7. In contrast to
such implications of YCK1, YCK2, and YCK3 in vesicular traffic, HRR25 was originally
proposed to carry out other important function(s). The HRR25
gene was identified by a mutation that conferred sensitivity to the
expression of HO. HO is a gene coding for a 65-kDa
endonuclease, which performs site-specific cleavage of double-stranded
DNA, and is essential for the initiation of mating-type
interconversion. Using a yeast strain harboring a galactose-inducible
HO gene, Hoekstra et al. (17) isolated mutants
that were unable to grow on galactose-containing medium. One of the
mutants, hrr25-1, showed sensitivity to continuous expression of the HO double-strand endonuclease, to
methylmethanesulfonate, and to x-ray irradiation. The
hrr25-1 mutant cells not only had a defect in DNA
double-strand break repair, but also showed poor sporulation, very slow
growth, and cell cycle delay in G2 (17).

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|
Fig. 7.
Genetic interactions between yeast CKIs and
components involved in vesicular transport. See "Discussion"
for details. AP-3, adaptor protein complex 3;
GCS1, a gene encoding GTPase-activating protein of
ARF; CHC1, the gene encoding clathrin heavy
chain.
|
|
Yck1p, Yck2p, and Yck3p have a motif of prenylation (GCC) in their C
termini, and in fact Yck2p is tightly associated with the plasma
membrane (29). Hrr25p does not have the prenylation motif, but was also
found exclusively in membrane fractions in differential centrifugation
analysis; HA-tagged Hrr25p cofractionated with plasma membrane and
nuclei (29). DeMaggio et al. (30) predicted that Hrr25p is a
multipotential protein kinase, generating phosphothreonine and
phosphoserine. They detected Hrr25p-dependent phosphorylation of nonspecific proteins found in the Hrr25p
immunoprecipitates by autoradiography. Hrr25p was also able to
phosphorylate tyrosine residues when the protein was expressed in
Escherichia coli (31). Ho et al. (13) reported
that Swi6p was phosphorylated by Hrr25p in their in vitro
kinase assay. Swi6p interacts with Swi4p to form the SBF (SCB
(Swi4/Swi6 cell cycle box)-binding factor) complex, which acts as a
transcription factor for some G1 cyclin genes, HO, and genes involved in transcriptional response to DNA damage.
HRR25 in Vesicular Transport--
We have shown in the present
study that rst2-1, an extragenic suppressor of
sec12-4, is a new mutant allele of HRR25
(hrr25-2). The hrr25-2 mutation not only
suppresses the ts growth of sec12-4, but in fact resumes
the secretion defect of sec12-4. The hrr25-2 mutant cells grow slowly; their doubling time is approximately 4-5
times longer than that of the wild-type. However, the resumed kinetics
of CPY transport is as fast as in the wild-type. Thus, besides the
functions in DNA damage repair and cell cycle control, Hrr25p must play
some role in the secretory transport pathway.
hrr25-2 harbors a mutation that causes the amino acid
replacement T176I. This residue is conserved among the four yeast CKIs and the mutant Hrr25 T176I protein with an HA tag showed little kinase
activity. We constructed another mutant allele of HRR25 that
causes the K38A replacement. This residue is also conserved in the
kinase domain, and the mutant protein was shown to have lost the kinase
activity. The overexpression of Hrr25p K38A also suppressed the ts
growth of sec12-4, although weakly. These results suggest
that it is the decrease of the kinase activity that is important for
Hrr25p to suppress sec12-4. However, the deletion of
HRR25, which is not a lethal event, did not suppress
sec12-4.
Two reasons can be considered to explain the difference between the
loss-of-function-type missense mutations (T176I and K38A) and the null
mutation. The hrr25 deletion is not lethal, but the disruptant cells are quite sick. They grow very poorly at 30 °C and
are almost inviable at high and low temperatures. Perhaps the null
mutant cells have so many problems to sustain growth, and even though
the loss of activity had a remedial effect on sec12-4,
other lesions may have concealed such suppression. The alternative,
more intriguing possibility is that the presence of the mutant protein
is required for the suppression. The observation that the
overproduction of Hrr25p T176I or K38A allowed the growth of the
hrr25 sec12-4 double mutant up to 35 °C supports this possibility.
Although the interaction between the active kinase and substrates may
be transient, a kinase-inactive mutant may form a stable protein-protein complex. In mitogen-activated protein kinase pathways, some substrates have been identified by two-hybrid screening with such
a kinase-minus mutant as a bait (32-34). It is possible that Hrr25p
T176I or K38A binds to its substrate(s) very tightly and, by doing so,
releases the budding block of sec12-4. There are some
differences between Hrr25p T176I and K38A. For example, the overexpression of Hrr25p K38A suppresses
hrr25 sec12-4
better than that of Hrr25p T176I, even though K38A appears to be more severely impaired in the kinase activity. This could be explained by
the positions of mutations; K38A is in the ATP-binding site and T176I
is in a putative substrate recognition site.
Target of Hrr25p in Vesicular Traffic--
Sec12p is not
phosphorylated under a normal condition of vegetative growth as far as
we examined by in vivo labeling
experiments,2 and is thus
unlikely to be a substrate of Hrr25p. This led us to postulate that the
target of the Hrr25p in vesicular transport is a negative regulator of
the Sec12p function. It could be directly regulating the function of
Sec12p or might transduce a signal to control vesicle budding
reactions. Then, what is this putative regulator? Sec31p, one of the
COPII components, was a good candidate because it had been shown to be
a phosphoprotein and the phosphatase treatment of Sec13p/Sec31p complex
inhibited vesicle budding (35). However, Sec31p was still
phosphorylated in the hrr25-2 mutant cells, and the
purified Sec13p/Sec31p complex was not subject to phosphorylation by
3HA-Hrr25p in our in vitro kinase assay.2 At the
moment, we have not yet been able to identify in vivo or
in vitro substrates of Hrr25p as candidates of the regulator of the Sec12p function. Considering the possibility that the titration of a regulator of Sec12p by the kinase-minus Hrr25p mutant (K38A or
T176I) is the cause of the sec12 suppression, this regulator might not necessarily be a substrate of the Hrr25p kinase.
The mutations in HRR25 could also invoke vesicle budding
from the ER in sec12-4 cells by bypassing the requirement
of Sec12p or COPII components. However, hrr25-2 does not
suppress the complete loss of Sec12p2 or the temperature
sensitivity of COPII mutants, such as sec13-1 and
sec23-1. Furthermore, although hrr25 K38A
suppresses sec12-4, it rather aggravates the temperature
sensitivity of some mutations in COPII, such as sar1-2 and
sec23-1. Specific suppression of sec12-4 by
hrr25-2 implies again the important role of Sec12p in the
regulation of vesicle budding. Our further work on the target(s) of
Hrr25p will help unveil the mechanisms of how the earliest event of
vesicle budding is controlled in living cells.
 |
ACKNOWLEDGEMENTS |
We are grateful to Howard Riezman (University
of Basel, Basel, Switzerland) for the anti-Gas1p antibody, Randy
Schekman (University of California, Berkeley, CA) for the
Sec13p/Sec31p complex and the anti-Sec31p antibody, Hiroshi Qadota
(Nara Institute of Science and Technology, Nara, Japan) for a plasmid,
and the members of the Nakano laboratory for helpful discussions.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid from the
Ministry of Education, Science and Culture of Japan and by a fund from
the Biodesign Project of RIKEN.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.
§
Supported by a postdoctoral fellowship from RIKEN and by a research
grant from the Human Frontier Science Program Organization.
To whom correspondence should be addressed. Fax:
81-48-462-4679; E-mail: nakano{at}postman.riken.go.jp.
The abbreviations used are:
ER, endoplasmic
reticulum; CKI, casein kinase I; CPY, carboxypeptidase Y; GEF, guanine
nucleotide exchange factor; HA, hemagglutinin; ts, temperature-sensitive; DTT, dithiothreitol; ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide gel
electrophoresis; FOA, 5-fluoroorotic acid; kb, kilobase pair(s) .
2
A. Murakami, unpublished data.
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