Commonly used Saccharomyces cerevisiae strains (e.g. BY4741, W303) are growth sensitive on synthetic complete medium due to poor leucine uptake

CommonlyusedSaccharomyces cerevisiae strains(e.g. BY4741,W303)are growth sensitive on synthetic completemediumdue to poor leucineuptakeRuth Cohen & David Engelberg

The Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

Correspondence: David Engelberg, The

Department of Biological Chemistry, The

Alexander Silberman Institute of Life Sciences,

The Hebrew University of Jerusalem,

Jerusalem 91904, Israel.

Tel.: 1972 26584718; fax: 1972 26584910;

e-mail: [email protected]

Received 10 December 2006; revised 8 March

2007; accepted 7 May 2007.

First published online 16 June 2007.

DOI:10.1111/j.1574-6968.2007.00798.x

Editor: Linda Bisson

Keywords

leucine uptake; leucine auxotrophicity; BAP2 ;

TAT1 ; LEU2 .

Abstract

It is reported that some of the widely used laboratory strains of Saccharomyces

cerevisiae (e.g. W303, BY4741) are sensitive to synthetic media containing all 20

amino acids [e.g. synthetic complete (SC) medium or supplemented minimal

medium]. To determine the molecular basis for this unexpected sensitivity,

a genomic library was screened and three genes were identified that, when

overexpressed, rescue cells from this phenotype. Two of the ‘rescuing’ genes,

BAP2 and TAT1, are related to transport of leucine, and one, LEU2, to synthesis of

leucine, showing that sensitivity to SC medium is associated with the leu2

mutation. The sensitive strains seem incapable of transporting leucine when

grown on synthetic complete media. This effect of the leu2 mutation should be

taken into consideration when analyzing the results of genetic screens and other

experiments performed with these strains.

Introduction

Saccharomyces cerevisiae cells can utilize a large variety of

nitrogen sources for their metabolic needs. They are capable

of manifesting such flexibility because they activate different

transport mechanisms and different synthesis pathways

according to changes in the availability and types of nitrogen

sources (Forsberg & Ljungdahl, 2001; Wilson & Roach,

2002). For example, cells cease the de novo synthesis of

amino acids that are provided in the growth medium

(specific control) (Fink et al., 1982). In contrast, many de

novo synthesis cascades are induced if even just one amino

acid exists at a low level (general control) (Hinnebusch,

1988). Low intracellular concentration of an amino acid is

represented by the accumulation of uncharged tRNAs that

are sensed by the Gcn2 kinase, which in turn activates the

Gcn4 transcriptional activator (Hinnebusch, 1993). The

authors have been studying the Gcn4 system, particularly

its activation by the Ras/cAMP pathway (Engelberg et al.,

1994; Marbach et al., 2001). As part of this study, it was

aimed to grow yeast cultures on media supplemented with

all amino acids in order to maintain low levels of Gcn4

activity. It was therefore attempted to grow several labora-

tory strains on media containing all 20 amino acids [syn-

thetic complete (SC) or supplemented minimal medium

(SMM), see ‘Materials and methods’]. It was found that

several laboratory strains, including some that are most

widely used for basic research, are sensitive to these condi-

tions. It was of interest therefore to reveal the molecular/

genetic basis for this phenomenon.

Materials and methods

Yeast strains and growth condition

The yeast strains are described in Table 1. Yeast–peptone–

destrose (YPD), synthetic dextrose (SD) minimal medium,

SC medium and SMM were prepared according to the CSH

manual (Kaiser et al., 1994). The YPD medium is composed

of 2% glucose, 1% yeast extract and 2% bacto peptone. The

SD medium is composed of 0.17% yeast nitrogen base

without amino acid and ammonium sulfate, 0.5%

NH4(SO4)2, 2% glucose and the required amino acids and

nitrogen bases. Yeast extract, peptone and y-nb w/o amino

acid and ammonium sulfate were purchased from Difco.

The SC medium is identical to the SD minimal medium, but

contains all amino acids at a concentration of 76 mg L�1.

This medium was prepared using a yeast synthetic drop-out

FEMS Microbiol Lett 273 (2007) 239–243 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

medium supplement without histidine, leucine, tryptophan

and uracil (Sigma Y2001). Each of the missing amino acids

and uracil was added to final concentrations of 40 mg L�1.

SMM is similar to SC, except that concentrations of most

amino acids are 20 mg L�1 and some are higher (Garrett,

1989).

Genetic screen

The DNA of a 2 m base genomic library was introduced into

SL5227LH (Stanhill et al., 1999) cells according to Schiestl &

Gietz (1989). The library was obtained from the laboratory

of G.R. Fink from the Whitehead Institute. The library was

prepared from DNA of the S288C background. Cells were

plated on SD-ura plates. Transformants were allowed to

grow for 2 days and then replica-plated to SC plates. From a

series of six transformations (providing about 8000 colonies

in each transformation), 40 colonies that grew on SC plates

were identified. Plasmid loss assays and re-transfection to

SL5527LH cells showed that 12 of the colonies were true

positives. Sequence analysis of plasmids isolated from these

colonies showed that some of the colonies contained the

same library plasmid. Altogether, the colonies yielded three

different plasmids, each capable of rescuing the sensitivity to

SC medium. The plasmids were found to contain the

following genomic fragments:

(1) Plasmid 1: Chromosome 2: 369412-376676.



Results

It was aimed wished to grow wild-type strains on media

containing all 20 amino acids (SC medium) in order to

maintain low levels of Gcn4 activity. Surprisingly, it was

found that three of the strains that were used, BY4741

(Brachmann et al., 1998), W303 and SL5527LH (see Table

1), were sensitive to this medium. Cells of the BY4741 and

SL5527LH did not grow even after a long period of incuba-

tion (72–96 h, Fig. 1). Cells of the W303 strain did grow, but

very slowly (Figs 2 and 3). Cells of the H4 strain grew on the

SC plates, but rather poorly (Fig. 1). All strains grew well on

either YPD or SD medium, supplemented with the missing

amino acid His, Leu, Trp, Lys, Met and Ura (40 mg L�1). The

same growth sensitivity was obtained when these strains

were plated on SMM plates (data not shown) (Kaiser et al.,

1994). SMM also contains all 20 amino acids, but most of

them at lower concentrations compared with the SC med-

ium (Garrett, 1989). Having realized that important and

widely used strains are sensitive to the SC medium, the

mechanistic basis of the phenomenon was sought. It was

first suspected that the high concentration of one particular

amino acid was toxic to these strains. Therefore, the growth

of the sensitive strains was tested on various SC media, each

lacking just one amino acid. Omission of any single amino

acid did not support the growth of the sensitive strains (data

not shown). It was concluded that these yeast strains are

unable to cope with increased concentrations of many

amino acids and are not sensitive to a particular one. Next,

genes were screened that, when overexpressed, could rescue

this phenotype. A 2 m library was introduced into SL5527LH

Strains: YPD SC

ΣL5527

ΣL5527LH

BY4741

SP1

H4

Fig. 1. Some yeast strains are sensitive to

synthetic media containing all amino acids

(SC medium). Indicated strains were grown to the

logarithmic phase on YPD media, serially diluted

and plated on plates supplemented with either

YPD (left) or SC (right). Plates were incubated at

30 1C for 72 h.

Table 1. Yeast strains used in this study

Strain Relevant genotype Source or reference

SP1 MATa his3 leu2 ura3 trp1 ade8

canr

M. Wigler (Engelberg

et al., 1994)

SL5227 MATa ura3 trp1 G.R. Fink

SL5227LH MATa his3 leu2 ura3 trp1 Stanhill et al. (1999)

W303 MATa can1-100 ade2-1 his3-

11,15 leu2-3 trp1-1 ura3-1

Yeast genetic Stock

Center (Berkeley, CA)

H4 MATa leu2-3, 112, ura3-52 A. Hinnebusch

BY4741 MATa his3D1 leu2D0 met15D0

ura3D0

Brachmann et al. (1998)

FEMS Microbiol Lett 273 (2007) 239–243c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

240 R. Cohen & D. Engelberg

cells and tranformants that can grow on SC plates were

sought (see details in ‘Materials and methods’). Three

different genomic fragments were isolated that when over-

expressed enabled SL5227LH cells to grow on SC. Deletion

analysis of these fragments identified a single gene in each

one that is responsible for rendering SL5527LH cells capable

of growing on SC medium. The genes were as follows:

(1) BAP2, encoding a high-affinity leucine permease,

functioning as a branched-chain amino acid permease

involved in the uptake of leucine, isoleucine and valine

(Grauslund et al., 1995).

(2) TAT1, encoding an amino acid transporter for valine,

leucine, isoleucine and tyrosine, low-affinity tryptophan

and histidine transporter (Schmidt et al., 1994; Bajmoczi

et al., 1998; Regenberg et al., 1999).

(3) LEU2, encoding beta-isopropylmalate dehydrogenase,

which catalyzes the third step in the leucine biosynthesis

pathway (Andreadis et al., 1984). Because a strain of the

S1278b background was used in the screen, these three

genes were introduced into the W303 and BY4741 strains as

well and it was found that they conferred resistance to SC

medium in these strains too (Figs 2 and 3). The results of the

screen indicate that sensitivity to SC medium is associated

with defective leucine transport. The three strains that were

sensitive to SC medium carry the leu2 mutation. The finding

that overexpression of BAP2 and TAT1 rescues the cells

strongly suggests that they are not able to take up external

leucine when grown on SC medium. Therefore, cells are

rescued either when leucine transport is enhanced, or when

their capability to synthesize leucine is resumed. To further

verify this possibility, a strain, isogenic to SL5527LH, but

bearing an intact endogenous single copy LEU2 gene was

tested. This strain, SL5527 (Table 1), was found to be

resistant to SC medium (Fig. 1). This indicates not only the

linkage between leucine auxotrophy and sensitivity to SC

medium but also that overexpression of the LEU2 gene is

not necessary to achieve resistance.

It is important to emphasize that all strains found to be

sensitive to SC medium are auxotrophic not only for leucine

but also for other amino acids (Table 1). Yet, their defect in

transporting external amino acids seems to be specific to

leucine.

Discussion

Yeast cells sense the availability of external sources of amino

acids through a battery of sensors. Based on the external

source available and the cell’s needs, cells choose to trans-

port some or all of the available amino acids. Both the

sensing and the transport are tightly regulated at the

transcriptional and posttranslational levels (Sophianopou-

lou & Diallinas, 1995; Stanbrough & Magasanik, 1995;

Forsberg & Ljungdahl, 2001; Omura et al., 2001). This

regulation promotes a high transport rate of amino acids,

which serve as good nitrogen sources whereas transporters

of other amino acids, which may be less useful, could be

down-regulated in some media. There must be a system,

however, that ensures transport of amino acids that are

essential to auxotrophs. Namely, a strain auxotrophic for a

given amino acid should be able to absorb it even if this

amino acid is not the preferred nitrogen source. In this

report, a case has been described in which this system fails. It

was demonstrated that some strains, when grown in the

presence of a good nitrogen source in addition to the full

scale of amino acids, reduce leucine transport, despite being

auxotrophic for leucine. This finding joins a list of previous

reports describing the conditions under which the systems

that should ensure transport of essential amino acids fail. It

has been reported, for example, that strains auxotrophic for

Trp and Tyr do not grow on media supplemented with

certain amino acids (Greasham & Moat, 1973). It was

proposed, and supported by direct measurements, that

amino acid uptake of the essential amino acid is defective

in these strains because the transport systems are saturated

by an overdose of amino acids in the medium (Greasham &

Moat, 1973). Other examples are the amino acid transport

mutants aat1-aat4. These mutants grow poorly on amino

acid-rich medium (YPD), but grow normally on minimal

Strains: YPD SC

BY4741

W303

BY4741+pLEU2 -2µ

W303+pLEU2 -2µ

ΣL5527LH

ΣL5527LH+pLEU2 -2µ

Fig. 2. Overexpression of the LEU2 gene allows

sensitive strains to grow on SC medium. Indicated

strains were transformed with pRS425 (2m-LUE2)

and grown to the logarithmic phase on SD-leu

media, serially diluted and plated on YPD (left) or

SC (right). Cells were incubated at 30 1C for 48 h

before scanning of plates.


241Yeasts are growth sensitive due to poor leucine uptake

medium. This phenotype was shown to depend on the

auxotrophy to leucine. An aat1 LEU2 strain grew normally

on rich medium (Garrett, 1989). It was proposed, therefore,

that aat1 cells are unable to take up enough leucine. The

second member of the aat family, aat2, is the ELM1 gene.

ELM1 codes for a serine–threonine protein kinase that acts

as an inhibitor of filamentous growth. An S288C elm1-15

trp1 strain is sensitive to stress in a similar manner to cells in

which the RAS/cAMP pathway is constitutively active. This

phenomenon is dependent on auxotrophy for tryptophan

(Garrett, 1997). Most importantly, auxotrophy to amino

acids was reported to be directly related to the ability of S.

cerevisiae to be pathogenic (Goldstein & McCusker, 2001;

Kingsbury et al., 2006). Goldstein et al. found for example

that leu2 mutants, of pathogenic isolates become severely

deficient in their pathogenic potential (Goldstein & McCus-

ker, 2001). Interestingly, in a study describing a plasmid-

dependent screen in laboratory strains carrying the ade3 and

leu2 mutations, it was concluded that parts of the screen’s

results were related to the fact that the strain used was

auxotrophic for leucine rather than to the question of

interest (Nigavekar & Cannon, 2002). In fact, in screens

performed in this strain, on the basis of ‘loss’ of a 2 m ADE3

plasmid and red color sectoring, BAP2 was isolated repeat-

edly. The authors suggested that Bap2p is the only func-

tional leucine transporter on rich media (YPD) because

the low-specificity general amino acid permease Gap1p is

expressed only under nitrogen-poor conditions. This expla-

nation may also hold for the observation that overexpres-

sion of the two permeases BAP2 and TAT1 rescues leu2�wild

type in the screen. It does not explain, however, why the

sensitive strains do grow on YPD and cannot grow on SC

medium. The concentration of amino acids in YPD is higher

than in SC medium, but is not balanced as in SC medium.

Another possible explanation is that in YPD, the high

concentration of amino acids is balanced by other compo-

nents (e.g. peptides, lipids) that exist in the yeast extract.

The latter explanation is favored here because it was

observed that strains that did not grow on SC plates did

grow on modified SC plates that were supplemented with

1% yeast extract (data not shown). Adding peptone to SC

plates did not cause this effect. Thus, the effect of high

amino acid concentration is balanced in YPD medium by

components in the yeast extract that probably absorb some

of it.

It should be noted that not all laboratory strains that

carry a mutation in LEU2 manifest sensitivity to SC

medium. The SP1 (Engelberg et al., 1994) and H4 strains

for example are able to grow on SC medium (Fig. 1). The

leucine transport machinery of these strains is clearly less

sensitive to a high amino acid concentration.

The major purpose of this note is to bring to the attention

of researchers that some of the most commonly used

laboratory strains, including BY4741, which is the parent of

the knockout library, the TAP library (Ho et al., 2002) and

the GFP fusion library (Huh et al., 2003), are sensitive to

Strains: YPD SC SD-ura

BY4741+pRS426

BY4741+pRS426

W303+pRS426

W303+pRS426

W303+pBAP2 -2µ

BY4741+pTAT1 -2µ

BY4741+pBAP2 -2µ

W303+pTAT1 -2µ

ΣL5527LH+pRS426

ΣL5527LH+pRS426

ΣL5527LH+pTAT1 -2µ

ΣL5527LH+pBAP2 -2µ

Fig. 3. Overexpression of either BAP2 or TAT1 rescues sensitive strains from their sensitivity to SC medium. Indicated strains were transformed with

either pRS426 or with indicated genes and grown to the logarithmic phase on SD-ura medium, serially diluted and plated on plates supplemented with

either YPD (left), SC (plates at the center) or SD-ura medium (right). Cells were incubated at 30 1C for 48 h before scanning of plates.

FEMS Microbiol Lett 273 (2007) 239–243c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

242 R. Cohen & D. Engelberg

synthetic media containing all the amino acids. This phe-

nomenon is related to leucine uptake and is manifested as an

auxotrophic phenotype. The effect of the leu2 mutation is

reflected most probably not only in the auxotrophic pheno-

type but also in many other biochemical and molecular

activities in the cell. Therefore, it could have an influence on

experimental results of unrelated studies in particular on

genetic screens that use BY4741 and W303 or one of their

thousands of progeny. It may be preferable to restore Leu2

activity in such a strain before conducting any experiment. If

not doing so, the interpretation of such results should take

into account the effect of the leu2 mutation.

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243Yeasts are growth sensitive due to poor leucine uptake

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Commonly used Saccharomyces cerevisiae strains (e.g. BY4741, W303) are growth sensitive on synthetic complete medium due to poor leucine uptake