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. 13: 305–312 (1997)
Saccharomyces cerevisiae Cell Lysis Mutations cly5 and
cly7 Define Temperature-Sensitive Alleles of PKC1, the
Gene Encoding Yeast Protein Kinase C
Department of Microbial Molecular Biology, Bristol Myers Squibb Pharmaceutical Research Institute, Princeton,
NJ, 08543, U.S.A.
Received 25 July 1996; accepted 25 August 1996
A set of temperature-sensitive Saccharomyces cerevisiae mutants designated cly (for cell lysis) 1–8 because the cells
lyse at high temperature was isolated in a large screen for yeast temperature-sensitive mutations (Hartwell, 1967).
Here we report the isolation of two plasmids, containing inserts that complement both the cly5 and cly7 mutations.
DNA sequencing revealed that both of these inserts contain the gene encoding yeast protein kinase C (PKC1) (Levin
et al., 1990). Sequencing of the mutant alleles revealed that cly5 and cly7 contain distinct mutations separated by 194
base pairs. Consistent with this, the cly5 and cly7 ts alleles do not complement each other, and they are genetically
linked to PKC1 and to each other. Like other temperature-sensitive pkc1 alleles, the temperature-sensitive phenotype
is eliminated by growth in high osmotic strength media (Levin and Bartlett-Heubusch, 1992). (? 1997 by John Wiley
& Sons, Ltd.)
Yeast 13: 305–312, 1997.
No. of Figures: 2. No. of Tables: 1. No. of References: 13
  — Saccharomyces cerevisiae; cell lysis; PKC1; protein kinase; cell wall
The isolation of Saccharomyces cerevisiae
temperature-sensitive (ts) mutations that lysed at
the non-permissive temperature was reported by
Hartwell (1967). Eight S. cerevisiae strains each
containing one of these mutations (called cly for
cell lysis) were deposited in the Yeast Genetic
Stock Center culture collection. The mutations are
thought to define eight CLY genes designated
CLY1–8. Chromosomal loci were found for three
of these genes through genetic crosses (Hawthorne
and Mortimer, 1973).
Through selection for complementation of the
ts phenotype of cly5 and cly7, we isolated two
plasmids that contain overlapping inserts from a
S. cerevisiae gene library. The inserts contain only
the PKC1 gene. This gene encodes a protein with
homology to human protein kinase C (Levin et al.,
1990). S. cerevisiae strains containing deletion or
*Correspondence to: J. E. McCullough.
CCC 0749–503X/97/040305–08 $17.50
? 1997 by John Wiley & Sons Ltd
point mutations in PKC1 grow normally on isotonic media but lyse rapidly when osmotic support
is removed (Levin and Bartlett-Heubusch, 1992).
Yeast protein kinase C is the first of a kinasesignalling cascade consisting of five kinases that
are required for cell wall development (Irie et al.,
1993; Lee et al., 1993). Three of these kinases are
related to the mitogen-activated protein kinases of
mammalian cells (Irie et al., 1993; Lee et al., 1993).
Another, encoded by the yeast BCK1 gene, is
directly activated by protein kinase C (Lee and
Levin, 1992).
A genetic cross using a strain marked at the
chromosomal location of our plasmid inserts
showed that PKC1 is closely linked to both cly5
and cly7. We show that the cly5 and cly7 alleles we
obtained from the YGSC are very closely linked
and do not complement. Both cly5 and cly7
mutant strains grow at the non-permissive
temperature on high osmotic strength media, a
characteristic of ts alleles of PKC1 (Levin and
.   . . 
Table 1. Saccharomyces cerevisiae strains used in this study.
cly5 ade1 ade2 ura1 his7 lys2 tyr1 gal1 MATa
cly7 ura1 leu2 aro7 gal1 ade 2 lys1 his2
ade2 his3 leu2 trp1 ura3 MATá
ade2 leu2 ura3 lys1 trp1
This study
leu2 ura1 trp1 lys2 ade1 ade2
This study
Dr Rodney Rothstein
YGSC, Yeast Genetic Stock Center.
Figure 1. Cly5-1 and cly7-1 mutant strains grow on Y13 agar supplemented with 0·5 -NaCl at
37)C. Three agar plates were identically inoculated with 12 yeast strains, four containing cly7-1
(right), four containing cly5-1 (centre), and four containing cly4-1 (left). Two plates (right and
centre) contain Y13 agar+0·5 -NaCl and the other plate (left) contains Y13 agar without added
NaCl. The centre and left plates were incubated at 37)C, and the right plate was incubated at 25)C.
Bartlett-Heubusch, 1992). DNA sequencing of the
cly5 and cly7 alleles revealed that they contain
distinct mutations 194 base pairs apart in the
catalytic C-terminal domain of the protein.
Yeast strains and media
Table 1 lists the source and relevant genotypes
of the S. cerevisiae strains used in this study. The
cly5 mutant (254), the cly7 mutant (X3119-2B),
and the parental strain (A364A) were obtained
from the Yeast Genetic Stock Center (University
of California, Berkeley, CA, U.S.A.). Strains 254
and X3119-2B were crossed with W303-1A (provided by Dr R. Rothstein) to obtain SGY1301 and
SGY1300 respectively. These strains contain the
leu2-112 mutation required for transformation
with the library. Standard yeast media were used,
as described by Sherman et al. (1987).
? 1997 by John Wiley & Sons, Ltd
DNA sequence analysis
DNA sequences were determined using the dideoxy termination method with the Sequenase kit
(US Biochemical Corp., Cleveland, OH, U.S.A.)
or on an Applied Biosystem (Foster city, CA,
U.S.A.) automated sequencer. Computer-assisted
searches and analyses of nucleic acid and protein
sequences were performed with the Genetics
Computer Group sequence analysis software
package (Devereux et al., 1984) and the GenBankEMBL database.
Yeast transformation and genetics
Yeast transformation was performed using
spheroplasts according to the method of Hinnen.
Yeast genetic crosses and tetrad analyses were
done using standard yeast methods (Sherman
et al., 1987).
. 13: 305–312 (1997)
   
Figure 2(A).
Integration of URA3 at PKC1 chromosomal locus
URA3 was integrated at the PKC1 chromosomal locus by integrative transformation (OrrWeaver et al., 1983). The insert from pC701 was
subcloned into a yeast integrating vector (pRS306;
Sikorski and Hieter, 1989) adjacent to the yeast
URA3 gene. This vector was cut at the SpeI site
within the PKC1 gene to assure integration at the
chromosomal PKC1 locus. S. cerevisiae strain
W3031A (genotype: CLY5, CLY7, ura3-52) was
transformed with this plasmid. Transformants
were selected on media lacking uracil and have the
URA3 gene tightly linked to the chromosomal
locus of the plasmid insert.
Plasmids containing genes that complement cly5
and cly7 were isolated from a yeast gene library in
the S. cerevisiae–Escherichia coli shuttle vector,
pYEP351 (Hill et al., 1986), by selection of transformants of cly5 and cly7 mutants that grow at
the non-permissive temperature. Two plasmids,
pC701 and pC501, having overlapping inserts,
were isolated. These plasmids had genes that
reproducibly complemented both mutations.
DNA sequence was obtained from three short
regions of the inserts. A search of the GenBank
database revealed that these sequences are identical to sequences in PKC1. Comparison of the
? 1997 by John Wiley & Sons, Ltd
. 13: 305–312 (1997)
.   . . 
Figure 2(B).
restriction maps of the two plasmids with a restriction map of the PKC1 gene shows that pC701
contains the entire PKC1 coding sequence and that
pC501 contains all of the coding sequence except
0·8 kb encoding the amino-terminal 243 amino
acids of the protein.
Cly5 and cly7 may be alleles of PKC1, or it is
possible that PKC1 expressed on this multi-copy
vector suppresses cly5 and cly7. To determine that
the plasmid insert containing PKC1 is genetically
linked to the cly5 and cly7 mutations, the URA3
gene was inserted at the chromosome location
of the insert by integrative insertion (Orr-Weaver
et al., 1983) as described in Materials and
Methods. The URA3-containing strain that was
obtained was crossed with two different yeast
? 1997 by John Wiley & Sons, Ltd
strains, one containing cly5 (SGY1301) and the
other containing cly7 (SGY1300) and both containing ura3-52. Diploids were sporulated and
tetrads dissected. Of 130 spores analysed, only
one recombination was observed between cly7
and URA3. Of 98 spores analysed, only five
recombinations were observed between cly5 and
URA3. This extremely tight linkage indicates that
cly7 and cly5 are alleles of PKC1.
Yeast cells containing all known ts alleles of
PKC1 grow in media containing 0·5 -NaCl at the
non-permissive temperature, probably because
the high ionic strength results in less stress on the
weakened cell wall (Levin and Bartlett-Heubusch,
1992). This is also true for cly5 and cly7. Figure 1
shows four cly7- and four cly5-containing strains
. 13: 305–312 (1997)
   
Figure 2(C).
growing on Y13 plates supplemented with 0·5 NaCl at the non-permissive temperature. Four
strains, each containing another cell lysis mutation, cly4, do not grow under these conditions.
This is consistent with cly5 and cly7 being alleles of
If cly5 and cly7 are indeed mutations in the same
gene, they should not complement. A diploid
heterozygous for both cly5 and cly7 was constructed and did not grow at 37)C. Thus, there
is no complementation, indicating that the two
mutations are in the same gene. The diploid was
sporulated and tetrads were dissected. No recombinants that could grow at 37)C were observed
among 30 tetrads. If these mutations are not
identical they are closely linked.
? 1997 by John Wiley & Sons, Ltd
DNA containing the cly5 and cly7 alleles and
PKC1 from the parental strain A364a was amplified using the polymerase chain reaction and subsequently sequenced. There are four, presumably
strain-specific, differences between the PKC1 allele
of A364a and that determined by Levin et al.
(1990). There is a C in the A364a allele instead of
T at position "20, an A in the A364a allele
instead of G at position 124, and Ts in the A364a
allele instead of Cs at positions 193 and 946. None
of these changes alters the derived amino acid
sequence (Figure 2).
The cly5 and cly7 sequences each have one base
different from the A364a sequence. The cly5 allele
has a T instead of C at position 2871, and cly7 has
T in place of C at position 3065 of the PKC1
. 13: 305–312 (1997)
.   . . 
Figure 2(D).
sequence (Figure 2). Both of these cause changes in
the amino acid sequence. These are probably the
mutations causing the ts phenotype of the two
alleles. This demonstrates that the cly5 and cly7
mutations are distinct mutations within the PKC1
The cly5 mutation changes amino acid 958 from
threonine to isoleucine. This is in the region of
PKC1 that is homologous to the catalytic domain
of other c-kinases. It corresponds to position 175
in conserved kinase catalytic subdomain VI
described by Hanks et al. (1988). This subdomain
contains the kinase catalytic site (asp at 166)
(Hanks and Hunter, 1995). Kinases almost always
have a hydrophillic amino acid at position 175.
Isoleucine is not found at this position in active
? 1997 by John Wiley & Sons, Ltd
kinases (Hanks and Hunter, 1995). C-kinases other
than PKC1 have aspartic acid at position 175.
The cly7 mutation changes proline to serine at
position 1023 of PKC1. This corresponds to position 237 in conserved kinase catalytic subdomain
IX of Hanks et al. (1988). Subdomain IX forms an
á-helix that stabilizes the catalytic loop through
hydrogen bonding (Hanks and Hunter, 1995). The
proline at 1023 is very well conserved among
kinases. All C-kinases have proline at this position.
The cly mutations are reported to be in distinct
complementation groups with cly7 linked to ura1
on chromosome VII (Mortimer and Hawthorne,
1973). Data from subsequent crosses do not support the linkage of cly7 and ura1, and cly7 has
been dropped from recent maps (Brown, personal
. 13: 305–312 (1997)
   
Figure 2. DNA sequence and protein translation of PKC1 from strain GRF88 (Levin et al., 1990). Four strain-specific differences
between the GRF88 and A364A sequences are indicated at positions "20, 124, 193 and 946. The sequence changes caused by the
cly5 and cly7 mutations are shown at positions 2871 and 3065 respectively.
communication). We can think of no explanation
for the complementation discrepancy other than a
trivial one such as a strain mislabeling. The most
original source for these mutants at this time is the
genetic stock center. The cly5 and cly7 mutations
located there do not complement.
Cly5 and cly7 are distinct alleles of PKC1 with
mutations 194 nucleotides apart. Both mutations
are within the recognized kinase catalytic domain
(Hanks et al., 1988). PKC1 disruption mutants
fail to grow at any temperature when not osmotically supported. Since cly5 and cly7 mutants grow
at 25)C, their altered kinases must retain some
activity at this temperature which is lost at 37)C.
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. 13: 305–312 (1997)
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