A Saccharomyces cerevisiae Genome-Wide Mutant Screen for Altered Sensitivity to K1 Killer Toxin

Copyright 2003 by the Genetics Society of America

A Saccharomyces cerevisiae Genome-Wide Mutant Screen for AlteredSensitivity to K1 Killer Toxin

Nicolas Page,1 Manon Gerard-Vincent, Patrice Menard, Maude Beaulieu, Masayuki Azuma,2

Gerrit J. P. Dijkgraaf, Huijuan Li, Jose Marcoux, Thuy Nguyen, Tim Dowse,Anne-Marie Sdicu and Howard Bussey3

Biology Department, McGill University, Montreal, Quebec H3A 1B1, Canada

Manuscript received November 5, 2002Accepted for publication December 12, 2002

ABSTRACTUsing the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened

for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall �-glucan receptorand subsequently forms lethal pores in the plasma membrane. Mutations in 268 genes, including 42 ingenes of unknown function, had a phenotype, often mild, with 186 showing resistance and 82 hypersensitiv-ity compared to wild type. Only 15 of these genes were previously known to cause a toxin phenotype whenmutated. Mutants for 144 genes were analyzed for alkali-soluble �-glucan levels; 63 showed alterations.Further, mutants for 118 genes with altered toxin sensitivity were screened for SDS, hygromycin B, andcalcofluor white sensitivity as indicators of cell surface defects; 88 showed some additional defect. Thereis a markedly nonrandom functional distribution of the mutants. Many genes affect specific areas ofcellular activity, including cell wall glucan and mannoprotein synthesis, secretory pathway trafficking, lipidand sterol biosynthesis, and cell surface signal transduction, and offer new insights into these processesand their integration.

THE sequenced and analyzed Saccharomyces cerevisiae are involved in cell wall synthesis and regulation (Shahi-nian and Bussey 2000). Here we describe the resultsgenome has enabled a program of precise targetedof global screens of haploids and homozygous and het-gene disruption, resulting in a collection of mutant strainserozygous diploid mutants for altered K1 toxin sensi-deficient in each gene (Winzeler et al. 1999; Giavertivity.et al. 2002; see also: http://sequence-www.stanford.edu/

group/yeast/yeast_deletion_project/deletions3.html).Such a collection promotes the discovery of cellular

MATERIALS AND METHODSroles for genes by facilitating the characterization ofmutant phenotypes and allows a comprehensive exami- Strains and media: Wild-type strains were BY4742 (MAT�)nation of the genetic complexity of a phenotype. We and BY4743 (MATa/MAT�; Brachmann et al. 1998), excepthave used the S. cerevisiae gene disruption set to screen where noted in Figure 1B, which also presents some results

from strain SEY6210 (Robinson et al. 1988). Deletant strainsfor K1 killer toxin phenotypes. Toxin resistance haswere from the Saccharomyces Genome Deletion Consortiumbeen extensively studied by classical genetics, and many(Giaver et al. 2002) and are available at Research Genetics

genes have been identified. This toxin is encoded on (http://www.resgen.com/products/YEASTD.php3; see Tablethe M1 satellite virion of the L dsRNA virus of S. cerevisiae 1 for complete genotype descriptions). Haploid big1 and pkc1

mutants were obtained by dissection of the heterozygous dip-(Wickner 1996). Toxin sensitivity results from bindingloid strains on media supplemented with 0.6 m and 1.0 mof the protein to the cell surface and its subsequentsorbitol, respectively. To improve spore viability of pkc1 tetrads,action at the plasma membrane promoting a lethal loss1.0 m sorbitol was added during the zymolyase treatment of

of cellular ions (reviewed in Bussey 1991; Breinig et asci. Yeast were grown in standard YPD medium (Shermanal. 2002). Defects in the genes involved in these pro- 1991), unless otherwise stated. YPD/G418 medium, used to

pregrow the mutants for 18 hr on 2% agar plates, is made ofcesses may change cellular sensitivity to this toxin, andYPD supplemented with 200 mg/liter geneticin (GIBCO-BRL,known resistant mutants define genes whose productsGrand Island, NY). To test for drug sensitivity, YPD platescontained 25 or 50 �g/ml of calcofluor white, 30 or 80 �g/ml of hygromycin B, or 0.05% SDS.

K1 killer toxin assay: K1 toxin sensitivity was measured as1Present address: Institute of Biochemistry, Swiss Federal Institute offollows (for details see Brown et al. 1994). Yeast mutant strainsTechnology, Zurich CH-8093, Switzerland.(haploid MAT� as well as the homozygous and heterozygous2Present address: Department of Bioapplied Chemistry, Osaka Citydiploids) were pregrown for 18 hr at 30� on YPD/G418 inUniversity, 3-3-138 Sugimoto, Sumiyoshi-ku Osaka, 558-8585, Japan.parallel with corresponding wild types pregrown on YPD. To3Corresponding author: Department of Biology, McGill University,control for variation in toxin activity between experiments,1205 Ave. Docteur Penfield, Montreal, Quebec H3A 1B1, Canada.

E-mail: [email protected] three wild-type controls were incorporated into every batch

Genetics 163: 875–894 ( March 2003)

876 N. Page et al.

TABLE 1

Yeast strains

Strain Genotype Source

BY4742 MAT� his3�1 leu2�0 lys2�0 ura3�0 Brachmann et al. (1998)BY4743 MATa/MAT� his3�1/his3�1 leu2�0/leu2�0 Brachmann et al. (1998)

LYS2/lys2�0 MET15/met15�0 ura3�0/ura3�0Haploida As BY4742, orf�::kanMX4 Winzeler et al. (1999)Heterozygousa As BY4743, orf�::kanMX4/ORF Winzeler et al. (1999)Homozygousa As BY4743, orf�::kanMX4/orf�::kanMX4 Winzeler et al. (1999)SEY6210 MAT� leu2-3,112 ura3-52 his3-�200 Robinson et al. (1988)

trp1-�901 lys2-801 suc2-�9HAB880 As SEY6210 except mnn9::kanMX2 Shahinian et al. (1998)HAB900b As SEY6210 except f ks1::GFP-HIS3 Ketela et al. (1999)

a Indicates mutants obtained from the Saccharomyces Genome Deletion Consortium.b Haploid derived from TK103 strain.

of mutants tested (100–600 mutants/batch). Approximately Cell wall composition analysis: Total �-glucan analysis: Hap-loid strains used for alkali-insoluble �-glucan determinations1 � 106 cells were resuspended in 100 �l of sterile water, of

which 5 �l was used to inoculate 5 ml of molten YPD agar were MAT� sla1� and big1�, respectively, obtained or derivedfrom the Saccharomyces Genome Deletion Consortium (seemedium (1% agar, 0.001% methylene blue, and 1� Halvorson

buffered at pH 4.7) held at 45�. Sorbitol was supplemented Strains and media above) and compared to wild-type strainBY4742, while mnn9� (HAB880) and fks1� (HAB900) werefor big1 and pkc1 mutants, and for a wild-type control, as

described above. This medium was quickly poured into 60- � compared to parental strain SEY6210. Crude cell walls wereisolated and the levels of alkali-insoluble �-1,3-glucan and15-mm petri dishes and allowed to cool for 1 hr at room

temperature. Then 5 �l K1 killer toxin (1000� stock diluted �-1,6-glucan quantified as previously described (Dijkgraaf etal. 2002). The big1� mutant and the corresponding wild type1:10; Brown et al. 1994) was spotted on the surface of the

solidified medium. The plates were incubated overnight at were grown in medium containing 0.6 m sorbitol to provideosmotic support.18� followed by 24 hr at 30� (48 hr for slow growth mutants).

For each mutant showing a “killing” or “death” zone different Alkali-soluble �-1,6- and �-1,3-glucan analysis: Alkali-soluble�-1,6- and �-1,3-glucan immunodetection was performed asfrom wild type, a picture comparing the mutant and appro-

priate control was taken with the IS-500 Digital Imaging Sys- described by Lussier et al. (1998) and summarized here. Yeastwere pregrown on YPD/G418 for 18 hr at 30�, grown fortem, version 2.02 (Alpha-Innotech). Two measurements of

the killing zone were made with PhotoShop 4.0 and the aver- 24 hr at 30� in 10 ml YPD liquid, and harvested by a 10-mincentrifugation at 1860 � g. Cell pellets were washed with 5 mlage was saved in a database (FileMaker Pro 5.0) together with

the picture. Mutants with a killing zone �90% or �110% were of water and resuspended in 100 �l of water plus 100 �l ofglass beads. The cells were then subjected to five cycles ofretested up to four times to confirm the observed phenotype.

These percentages were determined as [(mutant killing zone vortexing for 30 sec, interspersed with 30-sec incubations onice. Total cellular protein of the lysate was determined withdiameter)/(wild-type killing zone diameter) � 100]. A subset

of mutants showing killing zones �75% or �115% was selected the Bradford assay (Bradford 1976; Bio-Rad, Mississauga,ON, Canada) prior to alkali extraction (1.5 n NaOH, 1 hr,for further characterization.

K1 toxin survival assay: To determine cell survival after 75�). A set of 1:2 serial dilutions of the alkali-soluble fractionswere then spotted on Hybond-C nitrocellulose membranetoxin treatment, 200 �l of a cell culture grown to log phase

in YPD pH 4.7 and adjusted to OD600 0.5 was incubated with (Amersham, Oakville, ON, Canada). The immunoblotting wasperformed in Tris-buffered saline Tween containing 5% non-50 �l toxin (1000� stock diluted up to 1:25) for 3 hr at 18�

on a labquake. Percentage of surviving cells was calculated fat dried milk powder using either a 2000-fold dilution ofaffinity-purified rabbit anti-�-1,6-glucan primary antibodyfollowing plating onto YPD agar after incubation with toxin

and counting colonies after 2 days at 30�. (Lussier et al. 1998) or a 1000-fold dilution of anti-�-1,3-glucan primary antibody (Biosupplies Australia Pty, Victoria,Drug phenotype assay: Drug sensitivity was determined by

spotting diluted cultures on plates containing various drugs Australia), both with a 2000-fold dilution of horseradish perox-idase goat anti-rabbit secondary antibody (Amersham). Theas described (Ram et al. 1994; Lussier et al. 1997). Briefly, 5

ml of liquid YPD medium, inoculated with freshly grown cells membranes were developed with a chemiluminescence detec-tion kit (Amersham). Dot blots were scanned with a UMAXon YPD/G418, was incubated overnight at 30�. The cell density

of these exponentially growing cultures was standardized with Astra 1220s scanner and signals were quantitated with AdobePhotoshop software, using the histogram function. The levelwater at an OD600 of between 0.485 and 0.515, and 2 �l of a

set of 10-fold serial dilutions were spotted on YPD supple- of �-1,6- and �-1,3-glucan for each mutant was estimated bya comparison with a wild-type dilution series, with mutantsmented with calcofluor white, hygromycin B, or SDS (seeclassified by ranges of 20–35% (see footnote in Table 5).Strains and media for drug concentrations). Hypersensitivity

or resistance was monitored for each drug after 48 and 72 hrgrowth at 30�. The cells were also spotted on a control plate(YPD without drug), which allowed a comparison with the RESULTSgrowth rate of the mutants after 24 hr growth at 30�. Pictures

K1 toxin sensitivity of deletion mutants: The toxinof all conditions tested were downloaded into a FileMaker 5.0database (see above for details). sensitivities of deletion mutants for 5718 genes were

877K1 Toxin Phenotypes of Yeast Genes

compared to those of the parental strain. The screen Glucan synthesis: The yeast cell wall is made princi-pally of four components: mannoproteins, chitin, �-1,3-was performed on haploid (MAT�) and homozygous

diploid mutants, with toxin sensitivity being almost iden- glucan, and �-1,6-glucan (Orlean 1997; Lipke andOvalle 1998). Protein mannosylation and �-1,6-glucantical in both backgrounds. The heterozygous diploid

collection was also tested, with the finding that 42 genes synthesis defects are known to lead to toxin resistanceby altering the cell wall receptor for the toxin (see Shah-have a haploinsufficient toxin phenotype. The individ-

ual deletion of most genes has no effect on toxin sensitiv- inian and Bussey 2000 for a review; Breinig et al. 2002).Many mutations resulting in resistance to the K1 toxinity. These aphenotypic mutations include genes with a

wide range of other phenotypes, such as slow growth have a reduced amount of �-1,6-glucan in the cell walland show slow growth or inviability depending on theand respiratory deficiency, and provide an important

control for trivial cellular alterations that might affect severity of the defect, and we anticipated finding newgenes affecting these processes. A complex pattern ofthe killing zone phenotype. For mutants in almost all

genes, despite some affliction, the killer phenotype is glucan phenotypes was found among the mutants exam-ined for alkali-extractable �-1,6- and �-1,3-glucan levels,wild type. Mutants in 268 genes (4.7%) have a pheno-

type distinct from wild-type toxin sensitivity, with 15 of with reduced or elevated amounts of one or both poly-mers found (Table 5). Of mutants in 63 genes withthese genes previously known to have such a phenotype

(Shahinian and Bussey 2000; de Groot et al. 2001). glucan phenotypes, 55 had effects on �-1,6-glucan levels,with the remaining 8 having �-1,3-glucan-specific alter-Tables 2–4 list these mutants in functional groupings.

A given gene is listed just once although some could ations. Of the 55 with �-1,6-glucan phenotypes, 40 alsohad some �-1,3-glucan phenotype, with 15 showing abe included in more than one category. Although the

phenotypes are significant and reproducible, most null �-1,6-glucan-specific phenotype. Principal findings areoutlined below.mutants have partial phenotypes. For example, among

155 haploid-resistant mutants, only 30 are fully resistant �-1,6-Glucan reduced: Mutants for five genes showingpartial toxin resistance had specific but partial alkali-at the toxin concentration used (Table 2). Toxin sensi-

tivity can be suppressed or enhanced in mutants, leading soluble �-1,6-glucan reductions. Among these was the�-1,3-glucan synthesis-associated gene FKS1, and thisto resistance or hypersensitivity. Toxin resistance, which

was always found as a recessive phenotype, is likely mutant also had reduced levels of alkali-insoluble �-1,3-glucan (Figure 1B; Table 5). The involvement of Fks1pcaused by a loss of function of some component needed

for toxin action. In hypersensitive mutants the mutation in both �-1,3- and �-1,6-glucan biogenesis has been stud-ied further (Dijkgraaf et al. 2002). Mutants in CNE1synthetically enhances toxin lethality and can be func-

tionally informative. Among the mutations resulting in encoding yeast calnexin have less �-1,6-glucan (Shahi-nian et al. 1998), and this is also a mutant phenotypea toxin phenotype, 42 were in uncharacterized open

reading frames (ORFs) of unknown function. Of these, of the uncharacterized gene YKL037W encoding a smallintegral membrane protein.3 were given a KRE (k iller toxin resistant) number, and

8 genes with hypersensitive mutants were called FYV �-1,6-Glucan reduced with altered �-1,3-glucan: big1mutants had greatly reduced levels of �-1,6-glucan and(function required for yeast v iability upon toxin expo-

sure) and given a number (Tables 2–4). an increase in �-1,3-glucan. BIG1 is a conditional essen-tial gene retaining partial viability on medium with os-Mutants for 118 genes with toxin phenotypes were

examined for altered sensitivity to SDS, calcofluor white, motic support (Bickle et al. 1998). Heterozygous big1/BIG1 diploids showed haploinsufficient toxin resistanceand hygromycin B as hypersensitivity or resistance to

these compounds is indicative of cell surface defects (Table 4), and haploid mutant cells grew very slowlyon medium containing 0.6 m sorbitol and were toxin(Lussier et al. 1997; Ross-Macdonald et al. 1999). Mu-

tants in 88 of these genes showed some additional phe- resistant (Figure 1A). Determination of the amount ofalkali-insoluble glucan in the cell wall of a big1 mutantnotype (Tables 2–4), independently suggesting that they

have some cell surface perturbation. As �-1,6-glucan is showed that the �-1,6-glucan was 5% of wild-type levels(Figure 1B). The amount of �-1,3-glucan in big1 mutantsthe primary component of the cell wall receptor for the

toxin, mutants in 144 genes with toxin phenotypes were increased, possibly through some wall compensatorymechanism. Elsewhere, we have extended work on theexamined for alkali-soluble �-1,6- and �-1,3-glucan levels

(Table 5) with 63 showing an altered level of one or both role of Big1p in �-1,6-glucan biogenesis (Azuma et al.2002).polymers. Genes previously identified as killer resistant

provide positive controls for this global screen (Tables Mutants for 13 genes had reductions in both alkali-soluble �-1,6- and �-1,3-glucan (Table 5), and three are2 and 4). A number of characterized genes not known

to have altered toxin sensitivity were found, suggesting described briefly below. smi1/knr4 mutants are resistantboth to the K1 toxin and to the K9 toxin from Hansenulathat they have roles in cell wall or surface organization.

Most mutants fall into a limited set of functional classes mrakii and have wall glucan defects and a reduced in vitroglucan synthase activity (Hong et al. 1994a,b). Smi1pand define specific areas of cellular biology, some of

which are described below (see also Tables 2 and 3). localized to cytoplasmic patches near the presumptive

878 N. Page et al.

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bin

din

gpr

otei

n(O

SBP)

77w

t72

SUR

4a

YLR

372W

Ster

olis

omer

ase,

fatt

yac

idel

onga

se83

wt

74L

CB

83YJ

L13

4WSp

hin

goid

base

-ph

osph

ate

phos

phat

ase,

puta

tive

regu

lato

r84

wt

84

5.Se

cret

ion

/en

docy

tosi

s(2

3ge

nes

)ER

V41a

YML

067C

CO

PII

ER

-Gol

give

sicl

epr

otei

n0

wt

0C

LC

1YG

R16

7WC

lath

rin

ligh

tch

ain

0w

t0

SS

SK

RE1

1aYG

R16

6WT

RA

PPII

Gol

give

sicu

lar

tran

spor

tpr

otei

n0

wt

0S

wt

SL

UV1

YDR

027C

Invo

lved

inpr

otei

nso

rtin

gin

the

late

Gol

gi0

wt

0SA

C1a

YKL

212W

Rol

ein

Gol

gifu

nct

ion

and

acti

ncy

tosk

elet

onor

gan

izat

ion

0w

t0

SS

SSH

E4a

YOR

035C

Req

uire

dfo

rm

oth

er-c

ell-s

peci

fic

gen

eex

pres

sion

and

for

endo

cyto

sis

0w

t0

SS

wt

SWA

2YD

R32

0CC

lath

rin

-bin

din

gpr

otei

nre

quir

edfo

rn

orm

alcl

ath

rin

fun

ctio

n0

wt

0S

SS

RC

Y1YJ

L20

4CF-

box

prot

ein

invo

lved

inen

docy

tic

mem

bran

etr

affi

c0

wt

0S

SS

SAC

2YD

R48

4WSu

ppre

ssor

ofac

tin

mut

atio

n,

invo

lved

inve

sicu

lar

tran

spor

t0

wt

NA

ERV4

6YA

L04

2WV

esic

ular

tran

spor

tbe

twee

nG

olgi

and

ER

38w

t42

wt

wt

wt

VPS7

4YD

R37

2CV

acuo

lar

prot

ein

sort

ing

53w

t70

SS

SVA

C8

aYE

L01

3WR

equi

red

for

vacu

ole

inh

erit

ance

and

vacu

ole

targ

etin

g55

7965

wt

wt

wt

CH

C1

YGL

206C

Cla

thri

nh

eavy

chai

n59

wt

0C

OD

3a

YGL

223C

Com

plex

wit

hSe

c34p

-Sec

35p

invo

lved

inve

sicl

etr

ansp

ort

toth

eG

olgi

59w

t69

SS

SYP

T6

YLR

262C

GT

P-bi

ndi

ng

prot

ein

ofth

era

bfa

mily

69w

t73

ERV1

4a

YGL

054C

Stro

ng

sim

ilari

tyto

D.

mel

anog

aste

rcn

ipr

otei

n71

wt

76C

OD

2YN

L04

1CC

ompl

exw

ith

Sec3

4p-S

ec35

pin

volv

edin

vesi

cle

tran

spor

tto

the

Gol

gi73

wt

73A

RL

3YP

L05

1WR

equi

red

for

tran

spor

tfr

omE

Rto

Gol

gian

dG

olgi

tova

cuol

es73

wt

80

(con

tinue

d)

880 N. Page et al.

TA

BL

E2

(Con

tinu

ed)

K1

kille

rto

xin

deat

hzo

ne

(%)

Cal

cofl

uor

Gen

en

ame

OR

FD

escr

ipti

onof

gen

epr

oduc

tH

aplo

idH

eter

ozyg

ous

Hom

ozyg

ous

wh

ite

Hyg

rom

ycin

BSD

S

VPS6

1aYD

R13

6CV

acuo

lar

prot

ein

sort

ing

78w

t67

VPS6

7a

YKR

020W

Vac

uola

rpr

otei

nso

rtin

g78

wt

68VP

S1YK

R00

1CM

embe

rof

the

dyn

amin

fam

ilyof

GT

Pase

s;va

cuol

arso

rtin

gpr

otei

n79

wt

68PE

P3a

YLR

148W

Vac

uola

rpr

otei

nso

rtin

g84

wt

57S

SS

END

3a

YNL

084C

Req

uire

dfo

ren

docy

tosi

san

dcy

tosk

elet

alor

gan

izat

ion

85w

t70

6.Pr

otei

ngl

ycos

ylat

ion

(13

gen

es)

CW

H41

YGL

027C

ER

gluc

osid

ase

I0

wt

0S

Sw

tG

DA

1YE

L04

2WG

uan

osin

edi

phos

phat

ase

0w

t0

SS

SR

OT

2a

YBR

229C

ER

gluc

osid

ase

II,

cata

lyti

csu

bun

it0

wt

0S

SS

YUR

1aYJ

L13

9CM

ann

osyl

tran

sfer

ase

0w

t0

OST

3YO

R08

5WO

ligos

acch

aryl

tran

sfer

ase

gam

ma

subu

nit

092

33S

SS

PMT

1YD

L09

5WM

ann

osyl

tran

sfer

ase

0w

t68

KR

E2YD

R48

3W�

-1,2

-Man

nos

yltr

ansf

eras

e0

wt

NA

CW

H8

YGR

036C

Dol

ich

ol-P

-ph

osph

atas

e0

wt

75S

SS

AL

G6

YOR

002W

Glu

cosy

ltra

nsf

eras

e53

wt

72S

wt

SA

LG

8YO

R06

7CG

luco

sylt

ran

sfer

ase

61w

t79

Sw

tS

MN

N5

aYJ

L18

6WPu

tati

vem

ann

osyl

tran

sfer

ase

64w

t81

AL

G5

YPL

227C

Dol

ich

ol-P

-glu

cose

syn

thet

ase

70w

t79

Sw

tS

PMT

2a

YAL

023C

Man

nos

yltr

ansf

eras

e77

wt

56S

wt

S

7.Pr

otei

nm

odifi

cati

onor

degr

adat

ion

(6ge

nes

)M

AK

10YE

L05

3CSu

bun

itof

Mak

3p-1

0p-3

1pN

-term

inal

acet

yltr

ansf

eras

e56

wt

73R

wt

SK

EX1a

YGL

203C

Car

boxy

pept

idas

e(Y

SC-�

)57

wt

70S

RS

DO

C1

YGL

240W

Com

pon

ent

ofth

ean

aph

ase-

prom

otin

gco

mpl

ex67

wt

80B

TS1

aYP

L06

9CG

eran

ylge

ran

yldi

phos

phat

esy

nth

ase

72w

t65

VPS2

7a

YNR

006W

Vac

uola

rpr

otei

nso

rtin

g-as

soci

ated

prot

ein

80w

t67

YPS7

YDR

349C

GPI

-an

chor

edas

part

icpr

otea

se83

wt

58S

SS

8.C

ell

wal

lor

gan

izat

ion

(8ge

nes

)K

RE1

aYN

L32

2CG

PI-a

nch

ored

plas

ma

mem

bran

epr

otei

n0

620

SS

SK

RE6

aYP

R15

9Wci

s-Gol

gigl

ucan

ase-

like

prot

ein

072

0S

SS

FKS1

aYL

R34

2W1,

3-�

-d-G

luca

nsy

nth

ase-

asso

ciat

edpr

otei

n0

850

SS

wt

CN

E1a

YAL

058W

Cal

nex

in,

regu

lati

onof

secr

etio

nan

dce

llw

all

orga

niz

atio

n0

wt

0S

SS

SMI1

aYG

R22

9C�

-1,3

-Glu

can

syn

thes

ispr

otei

n0

wt

0S

SS

SBE2

2a

YHR

103W

Sim

ilari

tyto

budd

ing

prot

ein

Sbe2

p77

9180

SCW

4YG

R27

9CG

luca

nas

ege

ne

fam

ilym

embe

r84

wt

72EC

M30

YLR

436C

Invo

lved

ince

llw

all

biog

enes

isan

dar

chit

ectu

re84

wt

73

(con

tinue

d)


TA

BL

E2

(Con

tinu

ed)

K1

kille

rto

xin

deat

hzo

ne

(%)

Cal

cofl

uor

Gen

en

ame

OR

FD

escr

ipti

onof

gen

epr

oduc

tH

aplo

idH

eter

ozyg

ous

Hom

ozyg

ous

wh

ite

Hyg

rom

ycin

BSD

S

9.M

itoc

hon

dria

l,re

spir

ator

y,an

dA

TP

met

abol

ism

(17

gen

es)

ILM

1YJ

R11

8CPo

ssib

lyin

volv

edin

mit

och

ondr

ial

DN

Am

ain

ten

ance

0w

t0

AT

P2YJ

R12

1WF1

F0-A

TPa

seco

mpl

ex,

F 1�

-sub

unit

0w

t65

IMG

2YC

R07

1CR

equi

red

for

inte

grit

yof

mit

och

odri

alge

nom

e0

wt

75N

AM

2YL

R38

2CL

euci

ne-

tRN

Alig

ase

prec

urso

r,m

itoc

hon

dria

l54

7879

OC

T1

YKL

134C

Mit

och

ondr

ial

inte

rmed

iate

pept

idas

e60

wt

79O

XA

1YE

R15

4WC

ytoc

hro

me

oxid

ase

biog

enes

ispr

otei

n65

wt

83D

IA4

YHR

011W

May

bein

volv

edin

mit

och

ondr

ial

fun

ctio

n70

wt

60M

RPL

8YJ

L06

3CR

ibos

omal

prot

ein

L17

,m

itoc

hon

dria

l70

wt

73M

GM

101

YJR

144W

Mit

och

ondr

ial

gen

ome

mai

nte

nan

cepr

otei

n70

wt

76M

RPL

38YK

L17

0WR

ibos

omal

prot

ein

ofth

ela

rge

subu

nit

,m

itoc

hon

dria

l71

wt

81M

RPL

33YM

R28

6WR

ibos

omal

prot

ein

ofth

ela

rge

subu

nit

,m

itoc

hon

dria

l72

wt

77M

MM

1YL

L00

6WR

equi

red

for

mit

och

ondr

ial

shap

ean

dst

ruct

ure

73w

t66

ISA

2YP

R06

7WM

itoc

hon

dria

lpr

otei

nre

quir

edfo

rir

onm

etab

olis

m73

wt

72M

RPL

27YB

R28

2WR

ibos

omal

prot

ein

YmL

27pr

ecur

sor,

mit

och

ondr

ial

74w

t74

IMG

1YC

R04

6CR

ibos

omal

prot

ein

,m

itoc

hon

dria

l81

wt

73A

TP1

5YP

L27

1WF1

F0-A

TPa

seco

mpl

ex,

F 1ep

silo

nsu

bun

it84

wt

55w

tS

SB

CS1

YDR

375C

Mit

och

ondr

ial

prot

ein

,in

volv

edin

the

asse

mbl

yof

cytb

clco

mpl

ex85

wt

81

10.

Un

grou

ped

gen

es(1

6ge

nes

)SO

D1

YJR

104C

Cop

per-

zin

csu

pero

xide

dism

utas

e0

wt

0U

TH

1aYK

R04

2WIn

volv

edin

the

agin

gpr

oces

s0

wt

0w

tw

tw

tD

RS2

YAL

026C

P-ty

peca

lciu

m-A

TPa

se0

wt

56w

tS

SN

PL3

YDR

432W

Nuc

leol

arpr

otei

n31

wt

0S

SS

LEM

3YN

L32

3WPr

otei

nw

ith

sim

ilari

tyto

Ycx1

p,m

utan

tis

sen

siti

veto

bref

eldi

nA

44w

t45

Sw

tS

GL

Y1a

YEL

046C

l-th

reon

ine

aldo

lase

,lo

wsp

ecifi

city

45w

t55

SS

SA

TS1

YAL

020C

�-T

ubul

insu

ppre

ssor

71w

t78

YTA

7YG

R27

0W26

Spr

otea

som

esu

bun

it74

wt

80D

FG5

YMR

238W

Req

uire

dfo

rfi

lam

ento

usgr

owth

,ce

llpo

lari

ty,

and

cellu

lar

elon

gati

on74

wt

80T

HP1

aYO

L07

2WH

ypot

het

ical

prot

ein

7670

0S

SS

CD

C50

YCR

094W

Cel

ldi

visi

oncy

cle

mut

ant

77w

t62

NEW

1YP

L22

6WM

embe

rof

the

non

tran

spor

ter

grou

pof

the

AB

Csu

perf

amily

77w

t66

LYS

7YM

R03

8CC

oppe

rch

aper

one

for

supe

roxi

dedi

smut

ase

Sod1

p78

wt

62FP

S1a

YLL

043W

Gly

cero

lch

ann

elpr

otei

n79

wt

65S

SS

UT

R1

YJR

049C

Ass

ocia

ted

wit

hfe

rric

redu

ctas

eac

tivi

ty84

wt

70PE

X12

YMR

026C

Req

uire

dfo

rbi

ogen

esis

ofpe

roxi

som

es—

pero

xin

84w

t72

(con

tinue

d)

882 N. Page et al.

TA

BL

E2

(Con

tinu

ed)

K1

kille

rto

xin

deat

hzo

ne

(%)

Cal

cofl

uor

Gen

en

ame

OR

FD

escr

ipti

onof

gen

epr

oduc

tH

aplo

idH

eter

ozyg

ous

Hom

ozyg

ous

wh

ite

Hyg

rom

ycin

BSD

S

11.

Gen

esof

curr

entl

yun

know

nfu

nct

ion

orpo

orly

char

acte

rize

d(1

9ge

nes

)B

UD

14YA

R01

4CPr

otei

nof

unkn

own

fun

ctio

n54

wt

47S

SS

YFR

043C

Hyp

oth

etic

alpr

otei

n55

wt

70w

tw

tw

ta

YKL

037W

Wea

ksi

mila

rity

toC

aeno

rhab

ditis

eleg

ans

ubc-

2pr

otei

n59

wt

67S

SS

aYG

L00

7WQ

uest

ion

able

OR

F63

wt

70a

YNL

213C

Hyp

oth

etic

alpr

otei

n67

wt

NA

RS

wt

PIN

4a

YBL

051C

Sim

ilari

tyto

Schi

zosa

ccha

rom

yces

pom

beZ

6656

8_C

prot

ein

70w

t70

YOR

154W

Sim

ilari

tyto

hyp

oth

etic

alA

rabi

dops

isth

alia

napr

otei

ns

71w

t84

YLR

270W

Stro

ng

sim

ilari

tyto

YOR

173w

71w

tN

AR

wt

wt

YNL

063W

Sim

ilari

tyto

S-ad

enos

ylm

eth

ion

ine-

depe

nde

nt

met

hyl

-tran

sfer

ase

73w

t76

MO

N2

YNL

297C

Un

know

nfu

nct

ion

,se

nsi

tive

tom

onen

sin

and

bref

eldi

nA

80w

t70

TO

S1YB

R16

2CPr

otei

nw

ith

sim

ilari

tyto

Aga

1p81

wt

66YE

R14

0WH

ypot

het

ical

prot

ein

82w

t66

SYS1

aYJ

L00

4CM

ulti

copy

supp

ress

orof

ypt6

82w

t74

YLL

007C

Hyp

oth

etic

alpr

otei

n83

wt

67EA

F6a

YJR

082C

Hyp

oth

etic

alpr

otei

n83

wt

76YD

R12

6WSi

mila

rity

toh

ypot

het

ical

prot

ein

YLR

246w

and

YOL

003c

84w

t66

YGR

263C

Wea

ksi

mila

rity

toEs

cher

ichi

aco

lilip

ase

like

enzy

me

84w

t68

KR

E27

YIL

027C

K1

toxi

nre

sist

ance

phen

otyp

e;h

asa

hyd

roph

obic

dom

ain

8580

54YB

R28

4WSi

mila

rity

toA

MP

deam

inas

e85

wt

70

Mut

ants

not

liste

dar

eth

ose

wit

ha

wild

-type

phen

otyp

ein

hap

loid

orh

omoz

ygou

sdi

ploi

dba

ckgr

oun

dan

dth

ose

that

are

hyp

erse

nsi

tive

(Tab

le3)

orh

aplo

insu

ffici

ent

(Tab

le4)

.R

,re

sist

ant;

S,h

yper

sen

siti

ve;

wt,

wild

type

toC

alco

fluo

rw

hit

e,H

ygro

myc

inB

,an

dSD

S.N

A,

not

avai

labl

e.a

Indi

cate

sm

utan

tsw

ith

a�

-glu

can

phen

otyp

e(s

eeT

able

5).


TA

BL

E3

Gen

esw

hose

dele

tion

caus

eshy

pers

ensi

tivi

tyto

the

K1

kille

rto

xin

(hap

loid

orho

moz

ygou

sdi

ploi

dde

ath

zone

�11

5%of

the

wild

type

)

K1

kille

rto

xin

deat

hzo

ne

(%)

Cal

cofl

uor

Gen

en

ame

OR

FD

escr

ipti

onof

gen

epr

oduc

tH

aplo

idH

eter

ozyg

ous

Hom

ozyg

ous

wh

ite

Hyg

rom

ycin

BSD

S

1.K

inas

es,

phos

phat

ases

,si

gnal

tran

sduc

tion

(8ge

nes

)H

OG

1YL

R11

3Wse

r/th

rpr

otei

nki

nas

eof

MA

PKfa

mily

196

wt

160

wt

wt

SPB

S2YJ

L12

8CT

yros

ine

prot

ein

kin

ase

ofth

eM

AP

kin

ase

kin

ase

fam

ily19

3w

t17

9SP

S1YD

R52

3Cse

r/th

rpr

otei

nki

nas

e15

2w

tN

AST

E11

YLR

362W

ser/

thr

prot

ein

kin

ase

ofth

eM

EK

Kfa

mily

139

wt

NA

wt

wt

SSS

K1

YLR

006C

Tw

o-co

mpo

nen

tsi

gnal

tran

sduc

er13

5w

t11

1R

wt

SSS

K2

YNR

031C

MA

Pki

nas

eki

nas

eki

nas

eof

the

HO

Gpa

thw

ay13

0w

t11

4T

PD3

YAL

016W

ser/

thr

prot

ein

phos

phat

ase

2A,

regu

lato

rych

ain

A12

2w

tN

AD

IG1

YPL

049C

Dow

nre

gula

tor

ofin

vasi

vegr

owth

and

mat

ing

113

wt

118

Rw

tw

t

2.T

ran

scri

ptio

n(7

gen

es)

MED

2YD

L00

5CT

ran

scri

ptio

nal

regu

lati

onm

edia

tor

166

wt

NA

GA

L11

YOL

051W

RN

APo

lII

hol

oen

zym

e(S

RB

)su

bcom

plex

subu

nit

158

wt

162

SS

SSR

B5

YGR

104C

RN

APo

lII

hol

oen

zym

e(S

RB

)su

bcom

plex

subu

nit

131

wt

124

SS

SSR

B2

YHR

041C

RN

APo

lII

hol

oen

zym

e(S

RB

)su

bcom

plex

subu

nit

124

wt

128

ITC

1YG

L13

3WSu

bun

itof

Isw

2ch

rom

atin

rem

odel

ing

com

plex

121

wt

125

UM

E6YD

R20

7CN

egat

ive

tran

scri

ptio

nal

regu

lato

r11

8w

t13

9S

wt

SSW

16YL

R18

2WT

ran

scri

ptio

nfa

ctor

118

wt

119

SS

S

3.R

NA

proc

essi

ng

(6ge

nes

)PR

P18

YGR

006W

U5

snR

NA

-ass

ocia

ted

prot

ein

148

wt

103

wt

wt

wt

CB

C2

YPL

178W

Smal

lsu

bun

itof

the

nuc

lear

cap-

bin

din

gpr

otei

nco

mpl

exC

BC

139

wt

119

Rw

tw

tC

DC

40YD

R36

4CR

equi

red

for

mR

NA

splic

ing

137

wt

NA

SS

SN

SR1

YGR

159C

Nuc

lear

loca

lizat

ion

sequ

ence

bin

din

gpr

otei

n12

2w

t11

6S

wt

wt

BR

R1

YPR

057W

Invo

lved

insn

RN

Pbi

ogen

esis

120

wt

116

wt

wt

wt

STO

1YM

R12

5WL

arge

subu

nit

ofth

en

ucle

arca

p-bi

ndi

ng

prot

ein

com

plex

CB

C11

9w

t12

4

4.R

ibos

omal

and

tran

slat

ion

init

iati

onpr

otei

ns

(18

2

gen

es)

RPS

16B

YDL

083C

Rib

osom

alpr

otei

nS1

6.e

157

wt

116

RPS

19B

YNL

302C

40S

smal

lsu

bun

itri

boso

mal

prot

ein

S19.

e14

9w

tN

AR

PS24

BYI

L06

9C40

Ssm

all

subu

nit

ribo

som

alpr

otei

nS2

4.e

149

wt

144

RPS

24A

YER

074W

40S

smal

lsu

bun

itri

boso

mal

prot

ein

S24.

e12

9w

t14

2w

tw

tw

tR

PS10

AYO

R29

3WR

ibos

omal

prot

ein

S10.

e12

811

211

8R

wt

wt

ASC

1YM

R11

6C40

Ssm

all

subu

nit

ribo

som

alpr

otei

n12

7w

t13

0R

PS17

AYM

L02

4WR

ibos

omal

prot

ein

S17.

e.A

127

wt

128

wt

wt

wt

RPS

11A

YDR

025W

Rib

osom

alpr

otei

nS1

1.e

126

wt

139

RPS

23B

YPR

132W

40S

smal

lsu

bun

itri

boso

mal

prot

ein

S23.

e12

6w

t12

3

(con

tinue

d)

884 N. Page et al.

TA

BL

E3

(Con

tinu

ed)

K1

kille

rto

xin

deat

hzo

ne

(%)

Cal

cofl

uor

Gen

en

ame

OR

FD

escr

ipti

onof

gen

epr

oduc

tH

aplo

idH

eter

ozyg

ous

Hom

ozyg

ous

wh

ite

Hyg

rom

ycin

BSD

S

RPL

13B

YMR

142C

60S

larg

esu

bun

itri

boso

mal

prot

ein

124

wt

130

RPS

16A

YMR

143W

Rib

osom

alpr

otei

nS1

6.e

121

wt

135

wt

wt

wt

RPS

0BYL

R04

8W40

Sri

boso

mal

prot

ein

p40

hom

olog

B12

0w

t12

5R

PS30

BYO

R18

2CSi

mila

rity

toh

uman

ubiq

uiti

n-li

kepr

otei

n/r

ibos

omal

prot

ein

S30

120

wt

120

RPL

2BYI

L01

8W60

Sla

rge

subu

nit

ribo

som

alpr

otei

nL

8.e

118

111

136

RPS

4BYH

R20

3CR

ibos

omal

prot

ein

S4.e

.c8

117

wt

114

RPS

30A

YLR

287C

-A40

Ssm

all

subu

nit

ribo

som

alpr

otei

n11

6w

t11

4R

PL14

AYK

L00

6WR

ibos

omal

prot

ein

115

wt

145

wt

wt

wt

RPS

6BYB

R18

1CR

ibos

omal

prot

ein

S6.e

114

115

126

TIF

3YP

R16

3CT

ran

slat

ion

init

iati

onfa

ctor

eIF4

B15

4w

t13

9w

tw

tS

YIF2

YAL

035W

Gen

eral

tran

slat

ion

fact

oreI

F2h

omol

og12

2w

tN

A

5.Pr

otei

nm

odifi

cati

onor

N-g

lyco

syla

tion

(5ge

nes

)M

NN

9a

YPL

050C

Req

uire

dfo

rco

mpl

exN

-gly

cosy

lati

on15

2w

t14

6S

SS

AN

P1a

YEL

036C

Req

uire

dfo

rpr

otei

ngl

ycos

ylat

ion

inth

eG

olgi

135

wt

129

SS

SM

AP1

aYL

R24

4CM

eth

ion

ine

amin

opep

tida

se,

isof

orm

113

4w

t13

0S

SS

MN

N10

aYD

R24

5WSu

bun

itof

�-1

,6-m

ann

osyl

tran

sfer

ase

com

plex

133

wt

136

SS

SL

AS2

1aYJ

L06

2WR

equi

red

for

side

-ch

ain

addi

tion

toG

PI11

4w

t11

9

6.C

ellu

lar

pola

rity

(5ge

nes

)B

UD

30a

YDL

151C

K1

toxi

nh

yper

sen

siti

vity

phen

otyp

e13

4w

t10

7B

UD

22YM

R01

4WPr

otei

nw

ith

poss

ible

role

inbu

dsi

tepo

lari

ty12

8w

t14

4S

Sw

tB

UD

25YE

R01

4C-A

Prot

ein

invo

lved

inbi

pola

rbu

ddin

g12

3w

t11

5S

SS

BEM

1YB

R20

0WB

udem

erge

nce

med

iato

r12

1w

t11

4S

SS

BU

D27

aYF

L02

3WK

1to

xin

hyp

erse

nsi

tivi

typh

enot

ype

115

wt

157

7.N

ewFY

Vge

nes

(8ge

nes

)FY

V1a

YDR

024W

K1

toxi

nh

yper

sen

siti

vity

phen

otyp

e14

1w

t13

8FY

V4YH

R05

9WK

1to

xin

hyp

erse

nsi

tivi

typh

enot

ype

127

wt

116

FYV5

aYC

L05

8CK

1to

xin

hyp

erse

nsi

tivi

typh

enot

ype

125

wt

119

FYV6

aYN

L13

3CK

1to

xin

hyp

erse

nsi

tivi

typh

enot

ype

123

wt

112

FYV7

aYL

R06

8WK

1to

xin

hyp

erse

nsi

tivi

typh

enot

ype

120

wt

116

wt

wt

wt

FYV8

YGR

196C

K1

toxi

nh

yper

sen

siti

vity

phen

otyp

e;si

mila

rity

toA

rp1p

119

wt

121

SS

SFY

V10

aYI

L09

7WK

1to

xin

hyp

erse

nsi

tivi

typh

enot

ype

116

wt

115

wt

SS

FYV1

2a

YOR

183W

K1

toxi

nh

yper

sen

siti

vity

phen

otyp

e11

4w

t12

3w

tS

wt

(con

tinue

d)


bud site in unbudded cells and at the site of bud emer-gence (Martin et al. 1999) and may act in the polariza-tion of glucan synthetic components. CSF1 (YLR087C)encodes an integral membrane protein that may be aplasma membrane carrier. The null mutant is hypersen-sitive to K1 toxin, calcofluor white, SDS, and hygro-mycin; Tokai et al. (2000) showed the mutant to be saltand hydrogen peroxide sensitive with low temperaturedefects in growth and the uptake of glucose and leucine.LAS21 (YJL062W) participates in glycosylphosphatidylin-ositol (GPI) synthesis, adding an ethanolamine phos-phate to the �-1,6-linked mannose of the GPI mannosecore (Benachour et al. 1999). As this mannose core isthe site of attachment of the �-1,6-glucan moiety to GPI-linked cell wall proteins, altered levels of �-1,6-glucanmight be expected, although the basis of neither the�-1,3-glucan defect nor the mutant hypersensitivity toK1 toxin is evident, indicating a need for further work.

�-1,6-Glucan elevated: Killer mutants in 33 genes hadelevated levels of �-1,6-glucan (Table 5). A group of�-1,6-glucan overproducers are mutant in genes in-volved in assembly of the outer fungal-specific �-1,6-glucan chain of N-glycosyl chains (mnn9, mnn10, andanp1; see Table 5 and Figure 2). Mutants in these genesare hypersensitive to killer toxin and are described fur-ther in N-glycosylation below. A contrasting group of resis-tant mutants overproducing �-1,6-glucan (and to alesser extent, �-1,3-glucan) are in a subgroup of genesinvolved in cortical actin assembly and endocytosis (Ta-ble 2 and sla1 mutant in Figure 1). Our results areconsistent with work reporting thickened cell walls insome of these mutants (for a review see Pruyne andBretscher 2000). Cell wall synthesis is normally re-stricted to the growing bud, but in these mutants newmaterial is added inappropriately to the mother cell,resulting in a thickened wall (Li et al. 2002). It is surpris-ing that cells with thickened cell walls and more �-1,6-glucan can be killer toxin resistant, since resistance typi-cally arises through loss of cell wall �-1,6-glucan andless binding of the toxin. One explanation is that moretoxin is bound to the walls, reducing its effective concen-tration, a resistance mechanism proposed for the SMKTtoxin of Pichia farinosa (Suzuki and Shimma 1999). Asecond explanation is that the thickened cell wall blockstoxin access to the plasma membrane.

Mutants for other genes that specifically overproducealkali-soluble �-1,6-glucan have broadly acting geneproducts, with mutants expected to be pleiotropic andtheir effects indirect. These include MAP1 encodingone of an essential pair of methionine aminopeptidases;this mutant is killer toxin, calcofluor white, hygromycin,and SDS hypersensitive (Table 3) and has a randombudding pattern (Ni and Snyder 2001). ERG4 encodesan oxidoreductase required for ergosterol synthesis.This mutant is partially toxin resistant, hypersensitiveto calcofluor white, hygromycin, and SDS (Table 2),

TA

BL

E3

(Con

tinu

ed)

K1

kille

rto

xin

deat

hzo

ne

(%)

Cal

cofl

uor

Gen

en

ame

OR

FD

escr

ipti

onof

gen

epr

oduc

tH

aplo

idH

eter

ozyg

ous

Hom

ozyg

ous

wh

ite

Hyg

rom

ycin

BSD

S

8.U

ngr

oupe

dor

poor

lych

arac

teri

zed

gen

es(1

2ge

nes

)C

SF1a

YLR

087C

Req

uire

dfo

rn

orm

algr

owth

rate

and

resi

stan

ceto

NaC

lan

dH

2O2

140

wt

125

SS

SVI

D21

aYD

R35

9CM

utan

tim

pair

edin

fruc

tose

-1,6

-bis

phos

phat

ase

degr

adat

ion

139

wt

136

SS

SA

RV1

aYL

R24

2CL

ipid

and

ster

olm

etab

olis

m13

7w

t12

3S

SS

SEC

66a

YBR

171W

ER

prot

ein

-tran

sloc

atio

nco

mpl

exsu

bun

it13

3w

t14

4PF

D1

YJL

179W

Pref

oldi

nsu

bun

it1

133

wt

117

SS

SC

TF4

YPR

135W

DN

A-d

irec

ted

poly

mer

ase

�-b

indi

ng

prot

ein

127

wt

112

wt

wt

wt

SYG

1YI

L04

7CM

embe

rof

the

maj

orfa

cilit

ator

supe

rfam

ily12

6w

t14

5IW

R1

YDL

115C

Hyp

oth

etic

alpr

otei

n12

6w

t11

0YD

J1YN

L06

4CM

itoc

hon

dria

lan

dE

Rim

port

prot

ein

121

wt

130

APL

4YP

R02

9CG

amm

a-ad

apti

nof

clat

hri

n-a

ssoc

iate

dA

P-1

com

plex

116

wt

117

wt

Sw

tH

MO

1YD

R17

4WN

onh

isto

ne

prot

ein

115

wt

109

AD

K1

YDR

226W

Ade

nyl

ate

kin

ase,

cyto

solic

112

wt

127

SS

S

Mut

ants

wit

ha

wild

-type

phen

otyp

ein

hap

loid

orh

omoz

ygou

sdip

loid

back

grou

nd

and

thos

ew

ith

are

sist

antp

hen

otyp

ein

thes

eba

ckgr

oun

dsar

en

otlis

ted.

NA

,not

avai

labl

e.a

Mut

ants

wit

ha

�-g

luca

nph

enot

ype

(see

Tab

le5)

.

886 N. Page et al.

TA

BL

E4

Gen

esw

hose

dele

tion

resu

lts

ina

K1

kille

rto

xin

hapl

oins

uffi

cien

cyph

enot

ype

K1

kille

rto

xin

deat

hzo

ne

%C

alco

fluo

rG

ene

nam

eO

RF

Des

crip

tion

ofge

ne

prod

uct

het

eroz

ygou

sw

hit

eH

ygro

myc

inB

SDS

1.R

esis

tan

t(d

eath

zon

e�

90%

ofth

ew

ildty

pe;

28

3ge

nes

)R

SP5

YER

125W

Ubi

quit

in-p

rote

inlig

ase

59G

LC

7YE

R13

3Wse

r/th

rph

osph

opro

tein

phos

phat

ase

1,ca

taly

tic

chai

n69

PUP2

YGR

253C

20S

prot

easo

me

subu

nit

(�5)

70C

CT

4YD

L14

3WC

ompo

nen

tof

chap

eron

in-c

onta

inin

gT

-com

plex

72A

RC

35YN

R03

5CSu

bun

itof

the

Arp

2/3

com

plex

73C

CT

7YJ

L11

1WC

ompo

nen

tof

chap

eron

in-c

onta

inin

gT

-com

plex

78SM

D3

YLR

147C

Stro

ng

sim

ilari

tyto

smal

ln

ucle

arri

bon

ucle

opro

tein

D3

80w

tw

tw

tN

SA2

YER

126C

K1

toxi

nre

sist

ance

phen

otyp

e;n

ucle

arpr

otei

n81

AR

P3YJ

R06

5CA

ctin

-rel

ated

prot

ein

82A

RC

15YI

L06

2CSu

bun

itof

the

Arp

2/3

com

plex

83SF

H1

YLR

321C

Subu

nit

ofth

eR

SCco

mpl

ex83

wt

wt

wt

CC

T2

YIL

142W

Ch

aper

onin

ofth

eT

CP1

rin

gco

mpl

ex,

cyto

solic

84C

CT

5YJ

R06

4WT

-com

plex

prot

ein

1,ep

silo

nsu

bun

it84

TA

D3

YLR

316C

Subu

nit

oftR

NA

-spe

cifi

cad

enos

ine-

34de

amin

ase

85b

BIG

1aYH

R10

1CB

igce

llsph

enot

ype

85w

tw

tS

GC

D7

YLR

291C

Tra

nsl

atio

nin

itia

tion

fact

oreI

F2b,

43-k

Dsu

bun

it85

wt

wt

wt

SEC

53YF

L04

5CPh

osph

oman

nom

utas

e86

SSL

2YI

L14

3CD

NA

hel

icas

e86

AC

C1a

YNR

016C

Ace

tyl-C

oAca

rbox

ylas

e87

CD

C25

aYL

R31

0CG

DP/

GT

Pex

chan

gefa

ctor

for

Ras

1pan

dR

as2p

87w

tw

tw

tG

AA

1YL

R08

8WR

equi

red

for

atta

chm

ent

ofG

PIan

chor

onto

prot

ein

s87

wt

wt

wt

TA

F3YP

L01

1CC

ompo

nen

tof

the

TB

P-as

soci

ated

prot

ein

com

plex

87w

tw

tw

tT

IP20

YGL

145W

Req

uire

dfo

rE

R-to

-Gol

gitr

ansp

ort

87T

RS1

20YD

R40

7CW

eak

sim

ilari

tyto

Myo

1p87

wt

wt

wt

CEG

1YG

L13

0Wm

RN

Agu

anyl

yltr

ansf

eras

e(m

RN

Aca

ppin

gen

zym

e,�

-sub

unit

)88

MEX

67YP

L16

9CFa

ctor

for

nuc

lear

mR

NA

expo

rt88

wt

wt

wt

LSG

1YG

L09

9WR

equi

red

for

nor

mal

grow

th,

mor

phol

ogy,

mat

ing,

spor

ulat

ion

89ER

G27

YLR

100W

3-K

eto

ster

olre

duct

ase,

requ

ired

for

ergo

ster

olbi

osyn

thes

is89

wt

wt

wt

CD

C3

YLR

314C

Cel

ldi

visi

onco

ntr

olpr

otei

n89

wt

wt

wt

New

KR

Ege

nes

KR

E29

YER

038C

Tw

o-h

ybri

din

tera

ctio

nw

ith

Ym10

23p

and

Lys

14p

76K

RE3

3a

YNL

132W

Un

know

nfu

nct

ion

,A

mp1

p-in

tera

ctio

nco

mpl

ex(2

0m

embe

rs)

83

(con

tinue

d)


and has a random budding pattern (Ni and Snyder2001). ERV14 (YGL054C) and ERV41 (YNL067C) encodeCOPII vesicle coat proteins involved in endoplasmicreticulum (ER)-to-Golgi trafficking (Otte et al. 2001),and both show toxin resistance. Mutants in four genesof unknown function also overproduce alkali-soluble�-1,6-glucan (Table 5). Two of these genes, BUD27(YFL023W) and BUD30 (YDL151C), have random bud-ding patterns when mutated (Ni and Snyder 2001), andboth are hypersensitive to killer toxin. FYV5 (YCL058C)encodes a predicted small integral membrane protein,with the mutant sensitive to sorbitol and low tempera-ture (Bianchi et al. 1999) and K1 toxin hypersensitive.Finally, the null mutant of YGL007C has partial killertoxin resistance (Table 2).

N-glycosylation: Defects in N-glucosylation and its pro-cessing can lead to partial toxin resistance and reducedlevels of �-1,6-glucan (Romero et al. 1997; Shahinianet al. 1998). Our results extend this finding to manyother genes whose products are involved in the biosyn-thesis and elaboration of the Glc3Man9GlcNAc2 oligosac-charide precursor of N-glycoproteins (Tables 2 and 3;Figure 2). If Golgi synthesis of the fungal-specific �-1,6-mannose outer arm of the N-chain is blocked by muta-tion in OCH1 or in MNN9, MNN10, or ANP1 of themannan polymerase complex, toxin hypersensitivity re-sults, concomitant with higher levels of �-1,6-glucan inthe cell wall (Tables 3 and 5; Figures 1 and 2; andsee Magnelli et al. 2002 for mnn9). The glucan levelsobserved in an och1 mutant were similar to those ob-tained in a mnn9 mutant (not shown). To explore thisfurther we determined the alkali-soluble glucan levelsfor other mutants in the mannan polymerase complexand the outer chain �-mannosyltransferases (Figure 2),irrespective of toxin phenotype. A mutant in mnn11,part of the �-1,6-mannose-synthesizing mannan poly-merase complex, also showed elevated glucan levels, asdid mnn2 encoding the major �-1,2-mannosyltransferasethat initiates mannose branching from the �-1,6-glucanbackbone. However, a mutant in mnn5, whose geneproduct extends the �-1,2-mannose branches from the�-1,6-glucan backbone, had reduced levels of both�-glucans. Previous work showed that a small amountof glucan is attached to the N-chain structure (Tkacz1984; van Rinsum et al. 1991; Kollar et al. 1997), anda genetic study by Shahinian et al. (1998) also suggestedthis possibility. Our results show that core N-chain pro-cessing is required for wild-type �-1,6-glucan levels,while absence of the outer �-1,6-linked mannose sidechain or its first �-1,2-mannose branch can result in anincrease in cell wall �-1,6-glucan. However, mutants inlater mannosylation steps in elaborating branches fromthe outer �-1,6-linked mannose side chain have no effector lead to reduced �-glucan levels.

Lipid and sterol synthesis and ion homeostasis: Mu-tants for 10 genes involved in the biosynthesis or regula-

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tion of lipids or sterols show partial toxin resistance

888 N. Page et al.

TABLE 5TABLE 5

Genes whose deletion results in an altered alkali-soluble (Continued)�-glucan phenotype

Gene name ORF �-1,6-Glucan �-1,3-GlucanGene name ORF �-1,6-Glucan �-1,3-Glucan

Both reducedSMI1 YGR229C �� -1,6-Glucan only affectedPIN4 YBL051C �� ElevatedCSF1 YLR087C � �MAP1 YLR244C wtLAS21 YJL062W � �ANP1 YEL036C wtCOD3 YGL223C (�) ��ERG4 YGL012W wtPMT2 YAL023C (�) �ERV14 YGL054C wtARV1 YLR242C (�) (�)ERV41 YML067C wtMNN5 YJL186W (�) (�)KEX1 YGL240W wtFYV10 YIL097W (�) (�)FYV5 YCL058C wtKRE33 YNL132W (�) (�)BUD30 YDL151C wtOSH1 YAR044W (�) (��)BUD27 YFL023W wtSBE22 YHR103W (�) (��)YGL007W wtFYV6 YNL133C (��) (��)

Reduced�-1,6-Glucan elevated and �-1,3-glucan reducedYKL037W �� wt

MNN10 YDR245W ��CNE1 YAL058W � wtMNN9 YPL050C �FKS1 YLR342W � wtYUR1 YJL139C () (�)KRE11 YGR166W � wtSHE4 YOR035C () (��)PEP3 YLR148W (�) wt

�-1,6-Glucan reduced and �-1,3-glucan elevated�-1,3-Glucan only affectedBIG1 YHR101C �� ()ElevatedKRE1 YNL322C �� ()THP1 YOL072W wt KRE6 YPR159W �� ()YNL213C wt ROT2 YBR229C (�) ()BTS1 YPL069C wt ()

GPA2 YER020W wt ()Increase (I): , I � 100%; , 65 � I � 100; , 45 �FYV7 YLR068C wt ()

I � 65; (), 25 � I � 45; (), I � 25%. Decrease (D):��, 85 � D � 100; ��, 65 � D � 85; �, 45 � D � 65;Reduced(�), 25 � D � 45; (��), D � 25%.SEC66 YBR171W wt �

ACC1 YNR016C wt (�)SYS1 YJL004C wt (�)

cellular membrane potential leading to reduced toxin-�-1,6-Glucan and �-1,3-glucan affectedinduced ion permeability. Pertinently, defects in theBoth elevatedATP-dependent Drs2p and Atp2p membrane channelsEND3 YNL084C involved in cation and proton pumping confer toxinVRP1 YLR337C

SAC7 YDR389W resistance. The altered membrane composition in lipidLAS17 YOR181W () or sterol mutants could also affect secretory pathwayVAC8 YEL013W function, possibly linking their partial toxin resistanceFYV1 YDR024W phenotypes to those found in protein trafficking andIPK1 YDR315C ()

secretion (Table 2). For example, KES1 is implicated inFPS1 YLL043W ergosterol biology and can partially suppress the toxinSAC1 YKL212W resistance of a kre11-1 mutant, with Kre11p being in-VID21 YDR359C

EAF6 YJR082C volved in Golgi vesicular transport as a subunit of theCDC25 YLR310C () TRAPP II complex (Jiang et al. 1994; Sacher et al.GLY1 YEL046C () 2001).SUR4 YLR372W () High-osmolarity and stress response pathways: To sur-VPS61 YDR136C ()

vive hyperosmotic conditions, S. cerevisiae increases cel-UTH1 YKR042W () lular glycerol levels by activation of the high-osmolarityVPS27 YNR006W () glycerol (HOG) mitogen-activated protein kinase (MAPK)VPS67 YKR020W ()

FYV12 YOR183W () () pathway. Such activation leads to elevated transcriptionof genes required to cope with stress conditions, includ-(continued)ing the synthesis of glycerol with a resultant increase ininternal osmolarity (Posas et al. 1998; Rep et al. 2000).Mutants with an inactive HOG pathway are toxin hyper-(Table 2). These mutants have defects in membrane

structure, possibly affecting the efficiency of insertion sensitive, while deletion of protein phosphatases, suchas PTP3, PTC1, or PTC3, which act negatively on theof the toxin into the plasma membrane or altering the


A 10,000-fold reduction in cell viability was found whencompared to the wild type. Previous estimates indicatethat �3 � 104 molecules of toxin are required to kill awild-type cell (Bussey et al. 1979). We compared thesensitivity of the hog1 parental wild type from the dele-tion collection (strain BY4742) with strain S14a, onwhich the original lethal dose estimate was made, andfound the strains to be of similar sensitivity (data notshown). Thus, just a few toxin molecules per cell arerequired to kill a hog1 mutant, indicating that a func-tional HOG pathway provides cells with a powerful wayto ameliorate the effects of this toxin.

The sequence of action of the K1 toxin begins withits binding to �-1,6-glucan cell wall receptors (Shahi-nian and Bussey 2000). In a second step, the toxininserts into the plasma membrane in a receptor-depen-dent process (Breinig et al. 2002) and forms pores caus-ing the leakage of ions and cellular metabolites, leadingto cell death (Martinac et al. 1990; Ahmed et al. 1999).To explore the defect in a hog1 mutant we asked whereit occurred in the path of action of the toxin, by examin-ing its epistasis in double-mutant combinations of hog1with the toxin-resistant cell wall mutants kre1 and kre2,both of which block synthesis of the cell wall receptor.A kre1 hog1 mutant was as fully resistant as a kre1 singlemutant, and a kre2 hog1 mutant was nearly so. Thus,defects in the cell wall receptor preventing binding ofthe toxin are dominant over the hypersensitivity of thehog1 mutant. This result is consistent with hypersensitiv-ity occurring through some downstream effect such asion homeostasis and/or lethal pore formation. One con-sequence of the activation of the HOG pathway is the in-duced expression of the glycerol-3-phosphate dehydroge-

Figure 1.—Killer toxin sensitivity and quantification of ma-nase Gpd1p, required in glycerol biosynthesis (Albertynjor cell wall polymers of different strains. (A) A total of 5 �let al. 1994). To test whether impaired glycerol produc-of toxin was spotted onto agar seeded with a fresh culture of

each strain (see materials and methods). The mutant “kill- tion was the basis of the hog1 mutant hypersensitivity, aing zone” diameter was compared to the corresponding wild gpd1 gpd2 double deletion mutant was made to reducetype and expressed as a percentage (see materials and meth- glycerol synthesis (Garcıa-Rodriguez et al. 2000). Thisods). (B) Measurement of cell wall �-1,6- and �-1,3-glucan

mutant had wild-type toxin sensitivity (data not shown).levels was performed by extraction and fractionation of theseIn further efforts to identify the downstream effectorspolymers from cell wall preparations, followed by quantifica-

tion of the alkali-insoluble fractions. The haploid mutants of Hog1p responsible for the basal toxin resistance, wewere from the Saccharomyces Genome Deletion Consortium examined deletion mutants in the known transcription(sla1� and big1�) or from strains HAB880 and HAB900, re- factors of the pathway, namely Msn1p, Msn2p, Msn4p,spectively, for mnn9� and fks1� mutants (see Table 4). To

Hot1p, Sko1p, and Rck2p (Proft and Serrano 1999;facilitate comparison, the values of alkali-insoluble glucansRep et al. 1999, 2000; Bilsland-Marchesan et al. 2000).were expressed as percentages of the corresponding wild-type

level. The data represent averages of at least three indepen- All were wild type in sensitivity, as was the msn2 msn4dent experiments with standard deviations not exceeding double mutant.10%. Cell integrity signaling: In response to cell wall alter-

ations, S. cerevisiae stimulates the Mpk1/Slt2p MAP ki-nase by activation of a cell integrity signaling pathway

pathway, lead to resistance (Tables 3 and 2, respectively; under the control of PKC1 (Figure 3B). Loss of functionFigure 3B). Deletion of HOG1 resulted in a killing zone of this pathway results in deficiencies in cell wall con-diameter almost twice that of the wild type. For such struction and cell lysis phenotypes, which can be par-large killing zones, the diameter is limited by the diffu- tially suppressed by osmotic stabilizers (Levin andsion rate of the protein toxin and greatly underestimates Bartlett-Heubusch 1992; Paravicini et al. 1992; Roe-increased mutant sensitivity. To quantify sensitivity in a mer et al. 1994). Consistent with playing a key role inhog1 mutant, toxin-induced cell mortality was measured cell surface integrity, a pkc1 haploid mutant kept alive

by osmotic support is extremely sensitive to the toxin.using a cell survival assay (see materials and methods).

890 N. Page et al.

Figure 2.—Schematicsummary of N-glycan bio-synthesis in yeast. N-glycosylprecursor assembly is initi-ated in the endoplasmic re-ticulum. At the stage ofGlcNAc2Man9, three glu-cose residues are seriallytransferred from the Dol-P-Glc donor to the N-glycanby the glucosyltransferasesAlg6p, Alg8p, and Alg10p.Glucosylation is requiredfor efficient transfer of theN-glycan to target proteinsby a complex that includesOst3p. The glucose residuesare subsequently trimmedby the sequential action ofglucosidases I and II,Cwh41p and Rot2p, respec-tively. N-linked oligosaccha-rides undergo further matu-ration in the Golgi, whereaddition of the fungal-spe-cific “outer-chain” is initi-ated by Och1p and elabo-

rated by various enzymes, including the mannan polymerase complex (adapted from Orlean 1997; Shahinian and Bussey2000). Arrows indicate activation and bars indicate negative effects. (*) indicates essential genes; i.e., only heterozygous mutantswere tested. Genes whose deletion causes toxin hypersensitivity, red; resistance, blue; no phenotype, yellow; not tested, white.�-Glucans are shown as follows: �-1,6-glucan and �-1,3-glucan both reduced; �-1,6-glucan reduced and �-1,3-glucanwild type; �-1,6-glucan and �-1,3-glucan both wild type; �-1,6-glucan elevated and �-1,3-glucan wild type; �-1,6-glucan elevated and �-1,3-glucan reduced. Mnn2p, Alg10p, and Hoc1p are not listed in Tables 2 or 3; they are resistant orhypersensitive to K1 toxin, but fall outside of the chosen ranges.

However, most of the upstream activators of Pkc1p and duplicated gene mutants shows the phenotype (RPS0B,4B, 10A, 17A, 19B, 23B), suggesting that they have dis-all known downstream MAPK signaling components oftinct functions. Since some phenotypes were relativelythe cell integrity pathway show no toxin phenotype (seeweak (killing zone diameters �115% of the wild type),Figure 3B). The absence of phenotype for the upstreamnot all mutants are listed in Table 3. Of the 8 single-integral plasma membrane activators of the pathwaycopy genes of the small ribosomal subunit, heterozygousmay be explained by the functional redundancy of thedeletions in just 2 essential genes, RPS13 and RPS15,components (Verna et al. 1997; Ketela et al. 1999;gave toxin hypersensitivity (Table 4). The toxin hyper-Philip and Levin 2001). Rho1p, the GTP-binding pro-sensitivity phenotype was more prevalent among mu-tein involved in relaying the signal from the plasmatants in the small subunit (43%) than among those inmembrane to Pkc1p, is essential and the heterozygotethe large (16%). A total of 46 genes encode the largehas a wild-type phenotype. However, in the MAP kinaseribosomal subunit proteins, among which 35 are dupli-cascade downstream of Pkc1p, the kinase Bck1p andcated (Planta and Mager 1998); 12 of the duplicatedthe MAP kinase Mpk1p are unique and nonessentialgenes show toxin hypersensitivity when mutated (Tables(Levin and Errede 1995). The absence of a toxin phe-3 and 4).notype upon mutation of these components indicates

that hypersensitivity of a pkc1 mutant is not caused bythe absence of activation of the MPK1 MAP kinase path-

DISCUSSIONway, but in some other way (Figure 3B).Ribosomal subunit proteins: Defects in many ribo- The ability to directly establish a phenotype-to-gene

somal subunit proteins lead to toxin hypersensitivity. relationship is a great enabling strength of the mutantOf the 32 small ribosomal subunit genes, 8 are found collection. Moreover, since each gene can be examinedas single copy and 24 are duplicated, for a total of 56 simply by testing a mutant, partial or weak phenotypesORFs (Planta and Mager 1998). Toxin hypersensitivity can be readily analyzed (Bennett et al. 2001; Ni andis observed for mutants for 21 of the duplicated genes Snyder 2001). The collection allows comprehensive(Tables 3 and 4). A single deletion of either copy often screening and a knowledge of which genes have been

examined, overcoming many of the limitations of a clas-shows hypersensitivity. In some cases only one of the


sical random mutant screen. Despite the extensive use lection used here neither the haploid MATa or MAT�nor the diploid heterozygous or homozygous deletionof random screens for toxin resistance these failed to

saturate the genome, as we have found mutants in many of this gene had a phenotype. Thus, in this strain back-ground Tok1p has no detectable role in toxin action,new genes. In addition, the mutant collection allows

one to know which genes remain to be tested and, im- indicating that despite the ability of the toxin to activateconductance of Tok1p, this channel protein cannot beportantly, which genes do not have phenotypes. Such

comprehensive testing can turn up the unexpected, as the only target for the K1 toxin and is not a significantin vivo target in this sensitive strain. Having mutants inillustrated by a few examples. The extent of the relation-

ships between cell wall polymers was unanticipated. Wall all cellular pathways allows the pursuit of phenotypethrough functional modules and has value in makingglucan work normally focuses on one or the other glu-

can synthetic pathway, and these are implicitly seen to such connections. Some specific examples are discussedbelow.be specific. Yet fks1 mutants, defective for a component

of the �-1,3-glucan synthase, are affected for both �-1,3- Functional clustering: The screen identified severalexamples of interactions that connect biological func-and �-1,6-glucan (Figure 1A), as are a large number

of other mutants (Table 5). These interactions likely tions into larger cellular processes, sometimes alreadyknown in detail. For example, toxin phenotypes traceindicate synthetic or regulatory links between these poly-

mers. The mnn9 mutation, which blocks synthesis of the relationship between almost every biosynthetic stepof the N-glycosyl moiety of glycoproteins. The cytoskele-the outer �-1,6-mannose arm of N-glycans, was assumed

specific and has been used to simplify structural analyses tal mutants provide an example of a less well-character-of glucomannoproteins in the cell wall (Van Rinsum et ized connectivity. Here a set of mutants in cytoskeletalal. 1991; Montijn et al. 1994). The fact that a mnn9 processes has a common toxin resistance phenotypemutation has other secondary effects that increase the that correlates with mother cells showing abnormal wallamount of glucan in the wall is an unexpected complica- proliferation. This wall phenotype, which is not a gen-tion, with the possibility that previous work analyzed eral one for all cytoskeletal defects, has been reportedstructures absent from wild-type cells. Electrophysiologi- for individual genes (see Pruyne and Bretscher 2000).cal work links the Tok1p potassium channel with toxin This functional cluster of genes, which may function inaction (Ahmed et al. 1999). In the deletion mutant col- limiting wall growth to daughter cells, offers insight into

a new facet of morphogenesis.

Figure 3.—Schematic summary of signal transduction path-ways involved in osmoadaptive responses and cell wall synthesisin yeast. (A) Exposure to high extracellular osmolarity triggersan adaptive response mediated by two pathways that convergeat Pbs2p. One arm of the pathway involves the binding ofPbs2p to plasma membrane protein Sho1p. Pbs2p is phosphor-ylated by the Ste11p MAPKKK, through a process requiringCdc42p, Ste50p, and Ste20p (Desmond et al. 2000). A secondpathway involves the two-component osmosensor moduleSln1p-Ypd1p-Ssk1p, which activates Pbs2p via a pair of relatedMAPKKK proteins, Ssk2p and Ssk22p. Activation of this MAPKcascade culminates at Hog1p with Hog1p-dependent activa-tion of the Rck2p protein kinase and activation and inactiva-tion of transcription factors. The model also outlines the ac-tion of some negative regulators of the pathway (Posas et al.1998; Rep et al. 1999, 2000; Bilsland-Marchesan et al. 2000;and references therein). (B) Environmental stresses causechanges in cell wall state, which are detected by the Wscproteins and Mid2p and Mtl1p. The information is transmittedto Rho1p by the guanine nucleotide exchange factors Rom1pand Rom2p. Tor2p is also an activator of Rho1p, whereasSac7p and Bem2p are GTPase-activating proteins for Rho1p.Activated, GTP-bound Rho1p interacts with a transcriptionfactor (Skn7p) and regulates the activity of proteins involvedin cytoskeleton assembly (Bni1p), cell wall synthesis (Fks1p),and signal transduction (Pkc1p). Pkc1p in turn activates thecell integrity MAP kinase pathway and independently on “an-other arm” effects Rap1p-dependent transcriptional repres-sion of ribosomal protein genes (Li et al. 2000; Philip andLevin 2001; and references therein). For the color-codingscheme, see Figure 2.

892 N. Page et al.

The HOG pathway buffers toxin action: Mutants in high osmolarity (Rep et al. 2000), ASC1 had a significanthypersensitivity (Table 3). ASC1 encodes a 40S smallhog1 are close to being maximally sensitive to the toxin,

dying at �1 molecule/cell, while in a HOG1 strain, four subunit ribosomal protein, one of many small ribosomalprotein encoding genes that, when mutated, show toxinorders of magnitude more toxin is needed to kill a cell.

How is this HOG1-dependent resistance achieved? One hypersensitivity (see Table 3 and below). Together,these observations suggest that, if the phenotype ob-possibility is that the HOG pathway is stress induced as

the toxin causes ion loss. Activation of this signaling served in a hog1 mutant results from a defect in expres-sion, it is not through a single gene but may originatepathway may result in changes in membrane conduc-

tance, intracellular osmotic pressure, or some other from a combined deficiency in more than one gene.Signaling components involved in toxin sensitivity:stress response, which can act to reduce the efficiency

of the toxin in promoting loss of cellular ions. Although Although the HOG pathway is the only MAP kinasecascade showing a toxin phenotype, two upstream acti-the toxin sensitivity of a gpd1 gpd2 double mutant is

similar to wild-type cells, the possible involvement of vators of MAPK pathways were identified in the screen:SSK1 and PKC1. The toxin hypersensitivity of an ssk1Hog1p-dependent osmoadaptation cannot be excluded.

Consistent with this scenario, Garcıa-Rodriguez et al. mutant is consistent with its place upstream of the HOGsignal transduction cascade. However, no toxin pheno-(2000) observed increased intracellular glycerol levels

after treatment with the cell-wall-perturbing agent cal- type is found for the components of the cell integrityMAPK pathway signaling downstream of PKC1, namely,cofluor white, independent of the action of GPD1 and

GPD2. An alternative explanation that there is some the sequentially acting kinases Bck1p, the redundantpair Mkk1p and Mkk2p, and the Mpk1p MAP kinaseconstitutive HOG1-dependent effect on cell wall synthe-

sis seems less likely on the basis of the following observa- (Figure 3B). This raises the question of how Pkc1p sig-nals in producing a normal response to the toxin. Previ-tions. Epistatic tests using kre1 hog1 and kre2 hog1 mu-

tants are consistent with the HOG pathway acting at the ous genetic analysis suggested a bifurcation of the signal-ing downstream of PKC1 (Errede and Levin 1993;membrane or intracellularly, as cell wall mutants are

epistatic to the hog1 defect and remain toxin resistant Helliwell et al. 1998). Our data are consistent withsuch a model since some “other arm” of the PKC path-in double mutants. Deficiencies in the HOG pathway

result in extreme toxin sensitivity, and we reasoned that way, distinct from the Bck1p-dependent arm, is responsi-ble for the toxin phenotype. Additional evidence for anmutations in genes regulated by this pathway might also

cause hypersensitivity. In looking for candidates, it is alternative pathway comes from studies on the coordina-tion of cell growth and ribosome synthesis, where astriking that some components specific to the RNA poly-

merase II complex (e.g., Gal11p, Med2p, Rpb4p, Rpb3p, block in protein secretion reduces ribosomal proteingene transcription (Mizuta and Warner 1994; Nier-Rpb7p, Srb5p, and Srb2p) or components shared be-

tween RNA polymerases I, II, and III (e.g., Rpb8p, ras and Warner 1999). This mechanism is: (i) depen-dent on Pkc1p activity; (ii) not mediated by the cellRpc10p, and Rpo26p) all display a strong toxin hyper-

sensitivity, similar to that of HOG pathway mutants (Ta- integrity pathway MAPK cascade (BCK1 or MPK1); and(iii) blocked by rap1-17, a silencing-defective allele ofbles 3 and 4). Is this response specific to the HOG

pathway? Among the MAPK pathways in yeast (Hunter RAP1 (Li et al. 2000). We found that a heterozygous rap1mutant exhibits haploinsufficient toxin hypersensitivityand Plowman 1997; Gustin et al. 1998), only the HOG

pathway exhibits toxin hypersensitivity. Mutants in (Figure 3B), providing additional support for Rap1pbeing an effector of Pkc1p.SMK1, MPK1, and YKL161c, which encode, respectively,

the MAP kinase of the sporulation pathway, the cell Ribosomal subunit mutants show toxin sensitivity:The coupling of protein secretion to ribosome synthesisintegrity pathway, and a putative uncharacterized path-

way, are not toxin hypersensitive. Similarly, a null muta- through the PKC pathway (Nierras and Warner 1999;Li et al. 2000) raises the possibility of regulation op-tion in the MAP kinase kinase encoding gene STE7,

which is involved in both the haploid mating and inva- erating in the reverse direction: that is, defects in pro-tein synthesis mediated predominantly through 40S ri-sive pathways, has no effect on toxin sensitivity. These

observations suggest a possible connection between the bosomal subunit proteins might affect protein secretionand cell wall synthesis. The binding of the rough ERsignaling elements of the HOG pathway and the activity

of the RNA polymerase II complex. To investigate which ribosomes to Sec61p of the signal recognition particleis through the 60S ribosomal subunit (Beckmann et al.potential target genes of Hog1p are responsible for the

hypersensitivity, we looked for toxin phenotypes re- 1997), and fewer mutants in 60S ribosomal proteinshave toxin phenotypes, arguing that the coupling stepsulting from mutations in genes known to be induced

by osmotic shock (Rep et al. 2000). None of these genes in itself is unlikely to be the primary site of any sucheffect. A more mundane alternative explanation is thathave an effect comparable to a hog1 mutant. Similar

results were obtained for genes whose mRNA level is nonessential defects in protein synthesis through lossof redundant ribosomal proteins have nonspecificaffected by a mutation of HOG1. However, among the

genes whose mRNA level is diminished after a shift to knock-on effects on protein secretion/cell wall synthesis


F. Posas, 2000 Rck2 kinase is a substrate for the osmotic stress-through failure to make enough of a component re-activated mitogen-activated protein kinase Hog1. Mol. Cell. Biol.

quired for protein secretion. 20: 3887–3895.Brachmann, C. B., A. Davies, G. J. Cost, E. Caputo, J. Li et al., 1998Strength and limitations of comprehensive phenotyp-

Designer deletion strains derived from Saccharomyces cerevisiaeing with the collection: In addition to phenotypic cluster-S288C: a useful set of strains and plasmids for PCR-mediated

ing of genes, the simple discovery of biological roles for gene disruption and other applications. Yeast 14: 115–132.Bradford, M. M., 1976 A rapid and sensitive method for the quanti-genes through phenotype remains an important part

tation of microgram quantities of protein utilizing the principleof this screen. For example, a number of mutants inof protein-dye binding. Anal. Biochem. 72: 248–254.

poorly characterized genes have �-glucan phenotypes Breinig, F., D. J. Tipper and M. J. Schmitt, 2002 Kre1p, the plasmamembrane receptor for the yeast K1 viral toxin. Cell 108: 395–405.that warrant investigation. The yeast disruption mutant

Brown, J. L., T. Roemer, M. Lussier, A. M. Sdicu and H. Bussey,collection has limitations. Duplicated genes and gene1994 The K1 killer toxin: molecular and genetic applications

families having synthetic phenotypes but no phenotype to secretion and cell surface assembly, pp. 217–231 in MolecularGenetics of Yeast: A Practical Approach, edited by J. R. Johnston.when individually deleted will be overlooked. Also, theIRL Press/Oxford University Press, Oxford.1105 essential genes representing 18.7% of the yeast

Bussey, H., 1991 K1 killer toxin, a pore-forming protein from yeast.genome (Giaver et al. 2002) cannot be screened di- Mol. Microbiol. 5: 2339–2343.

Bussey, H., D. Saville, K. Hutchins and R. G. Palfree, 1979 Bind-rectly. Haploinsufficiency phenotypes in heterozygotesing of yeast killer toxin to a cell wall receptor on sensitive Saccharo-disrupted in one copy of an essential gene provide amyces cerevisiae. J. Bacteriol. 140: 888–892.

partial solution, as in the case of BIG1. In our screen de Groot, P. W. J., C. Ruiz, C. R. Vazquez de Aldana, E. Dueas,V. J. Cid et al., 2001 A genomic approach for the identificationsuch haploinsufficiency was found in the heterozygousand classification of genes involved in cell wall formation and itsmutants of 42 genes, but we still do not know the fullregulation in Saccharomyces cerevisiae. Comp. Funct. Genom. 2:

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GTPase and Ste20 PAK-like kinase regulate Sho1-dependent acti-essential genes would improve the value of the collec-vation of the Hog1 MAPK pathway. EMBO J. 19: 4623–4631.

tion for screening these genes. Dijkgraaf, G. J. P., M. Abe, Y. Ohya and H. Bussey, 2002 Mutationsin Fks1p affect the cell wall content of �-1,3- and �-1,6-glucan inWe thank Angela Chu and Ron Davis (Stanford University) andSaccharomyces cerevisiae. Yeast 19: 671–690.Sally Dow (Rosetta Inpharmatics) for providing strains and assistance.

Errede, B., and D. E. Levin, 1993 A conserved kinase cascade forWe also thank Ashley Coughlin, Steeve Veronneau, Marc Lussier, and MAP kinase activation in yeast. Curr. Opin. Cell Biol. 5: 254–260.Terry Roemer for discussions and contributions and Robin Green Garcıa-Rodriguez, L. J., A. Duran and C. Roncero, 2000 Cal-and Federico Angioni for comments on the manuscript. Supported by cofluor antifungal action depends on chitin and a functionaloperating and CRD grants from the Natural Sciences and Engineering high-osmolarity glycerol response (HOG) pathway: evidence for

a physiological role of the Saccharomyces cerevisiae HOG pathwayResearch Council of Canada. N.P. was a predoctoral fellow of theunder noninducing conditions. J. Bacteriol. 182: 2428–2437.Fonds pour la Formation de Chercheurs et l’Aide a la Recherche

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A Saccharomyces cerevisiae Genome-Wide Mutant Screen for Altered Sensitivity to K1 Killer Toxin

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