BIOCHEMICAL

Vol. 71, No. 4, 1976

DISCRIMINATION

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

BETWEEN CHOLESTEROL A?JD ERGOSTEROL BY YEAST MEMBRANES hy

John H. Adler, Bernard C. Sekula, and Kenneth R. Nes, Department of Biological Sciences, Drexel University Philadelphia, Pennsylvania 19104

William

June 25,

Received

Krevitz

1976 sulmnary

The typical sterol of animal membranes (cholesterol) failed to support normal growth of yeast under anaerobic conditions when compared to the growth induced by the organism's natural sterol (ergosterol). This patholow was evident in strongly reduced numbers of cells, failure of budded cells to separate, and premature death. This study demonstrates that one or all of the *5,7 -, AZ2 -, and 24B-methyl groupings present in erEostero1 are functionally significant. Introduction Substantial architectural also

components

known

to

ma1 kingdom dominant

is

be highly

reduction

is

of

mutation

generations, structure

;-.---.--

but

and a A

of the

could

would

sterol

play

Regulation

-sterols

occurs

22

-bond

is

without

be necessary

giving

ergosterol

originally

through

function.

in

the which Such bioarisen

succeeding

to assume a lot.7 correlation

membraneous

hand,

reduced,

having cbanoe

as the

is not

sterol.

is

in the ani-

cholesterol

of the animal

as

biosynthesis

On the other

introduced

transnitted

role

For instance,

system

fortuitous

principal

of sterol

to give

he entirely

and its

their

of the pathway.

derivative

and then it

1.

the A 5 , 7 -diene

the 7,22-bisdehydro-24B-methyl

by random

sterols

on phvlorZenetics.

A 5,7,24 end-product

methylated,

differences

that

of membranes

and Basidiomycetes

synthetic

%or

indicates

dependent

and functional

Ascomycetes A24-bond

evidence

In agreement

between with

the this

- ____.

a review of the evidence in biology as a whole see (l)., The specific role of sterols in fungi as constituents of membranes is supported by the isolation of ergosterol from the mitochondria of --NeurosDora.crassa (i) and the isolation of 24-dehydroergosterol from the protoplasmic membrane (3) of a veast strain, Saccharomyces cerevisiae N.C.Y.C. 3hh, containing .-24-dehydroeraosterol as the dominant sterol (4).

Copyright 0 I976 by Acudemic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 71, No. 4,1976

explanation

are

the

degree

of structural

support

anaerobic

question

in

specificity

for

growth

on the

observed'.

one or all natural

suppositions

maximally

the

This

indicates

of yeast between

A 22 -,

Substantial is

actually

a Xiszh

sterols

In order

either

to clarify

this

of cholesterol favoring

a functional

groups

which

information

and hiospnthetic

lacks

different

differences

and 24B-methyl

Yaterials

yeast

of the effects

and by inference,the

function

that

at al12.

study

there

RESEARCH COMMUNICATIONS

and that

or not

of yeast.

of the A5,7 -,

sterol

relationship

growth

literature

sterol

we have made a quantitative

ergosterol were

AND BIOPHYSICAL

and ergosterol

significance exist

supports

to

in the a causal

events.

and Yethods -

A wild type diploid Saccharo=es -- cerevisiae (ATCC 18790) was cultured .-~I__ under anaerobic conditions (which prevents sterol biosynthesis) in a synthetically compounded yeast nitrogen base described by Andreasen and Stier (9) which was modified by the inclusion of only the following vitamins: biotin, calcium pantothenate, nicotinamide, pyridoxine-HCl and thiamine-HCl. Sterols (20 mg/l) were added in a susnension of Tween SO (15 ml/l) which provided olei'c acid. Growth was measured microscopically as well as through the use of a Coulter Counter, Model TATI, equipped with a population accessory. Results When grown a stationary

on ergosterol,

phase

of -ca.

eq.uivalent

to 100 thousand

stationary

phase

were

single

and Discussion

yeast 100 million cells/ml entities,

increased cells/ml (Pig.

1A).

Tqhile

in numbers in

three

?Tearly

rapidly days

all

in the early

reaching

from an inoculum

of the cells phase

at

of growth

---

2 The idea of an "all-or-nothing" response originated with J. 14. Proudlock et al. (5) who found that various sterols were either "active" or "inactive" in their ability to support anaerobic growth of yeast. Subsequently, viable aerobic mutants with altered sterol pathways, e.g., deletion of the 24-methyl transferase, were obtained (6,7). In addition ergosterol requiring mutants are known, and in one case it is reported, though without publication of data, that, since stigmasterol, sitosterol, and cholesterol also support growth, there is no stringent requirement for ergosterol (8).

3 Our results are anticipated by the exploratory experiments of Andreasen and Stier (9) who state without further detail that cholesterol, while supporting growth of anaerobic yeast, did so "to a lesser extent than ergosterol". Structural effects have also been observed more recently (10) which demonstrate a definite decrease in both optimal and permissive growth tempera8(9) 522-ergostatures of a yeast mutant when ergosterol was replaced by A dien-3S-01. In addition, the sterol substitution affected the activity of membrane-bound enzymes. For related physical evidence see (11,12).

1297

Vol. 71, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

COUNT

,*-

-

-

CnOLc8Tf

TWELN CELL

llOL

l 0

COUNT