Heterogeneous changes in amino acid transport activity in E. coli lipid overproducing mutants

BIOCHEMICAL AND BJOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 50, No. 2,1973

HETEROGENEOUS CHANGES IN AMINO ACID TRANSPORT ACTIVITY

IN -_I E. COLI LIPID OVERPRODUCING MUTANTS Joseph

T. Holden',

Nedra

M. Utechl,

Christine

George

D, Hegeman*

and

N. Kenyon*

Medical Center, 'Division of Neurosciences, City o Hope National Duarte, California 91010, and h Department of Bacteriology and Immunology, University of California, Berkeley, California 94720

Received

November

9,1972

SUMMARY--Mutants of --E. coli that overproduce fatty acids or excrete phosphowdisplay heterogeneous changes in the transport rates for some but not all amino acids. Glutamic acid and proline are transported more rapidly than normal, asparagine and lysine are transported less rapidly. Transport rates for aspartic acid and glutamine are not altered. Only one of two kinetically distinct glutamate transport components has an elevated Vmax value. The findings suggest that individual amino acid transport catalysts are affected differently by the structural or metabolic changes evoked in these mutants,

terial

Several

groups

metirane

lipids

Most of these glycerol

studies

Amino

plantarum

rendered

deficient

media

effects

largely

acid

at least we wish alterations

of the for

the

hydrocarbon

few system

to report in the

that

studied

two lipid

activity

0 I973 by Academic Press, Inc. of reproduction in any form reserved.

13)

it

chain

amino 266

general

markedly

overproducing

of several

saccharide

transport

in Lactobacillus or biotinconcerning

on the emplacement is

so far.

can be con-

(J-JO).

some uncertainty

biosynthesis

Jy agreed

affects

As an extension mutants acid

display

transport

or

or glycerol

in pantothenate-

is

acid

composition

acids

in bacsystems.

fatty

a few inducible

there

(1, 3, 5-g,

acid

lipid

fatty

by growth

in lipid

of transport

also have been studied

changes

Although

of changes

defective

membrane

only

lipid-deficient

fatty

with

unsaturated

transport

catalysts

transport

mutants

involved

(11-13).

the effects

and activity

in which

supplied

of an interruption

nature

Copyright AlI rights

utilized

enzymes

have

systems.

brane

have

by exogenously studies

have explored

on the synthesis

biosynthetic

trolled These

recently

the

of memthat

transport

the activity

of such studies, divergent systems.

VoL50,No.

BIOCHEMICAL

2, 1973

and Methods

Materials

cultivation

The isolation, strains tal

are described

strain,

is

Mutant

E-20

E-52L

excretes

cultures

contains

0.6

The cells lacking

grown

were

mg/ml

amino

oxyacetic

medium acid

acid.

filters

histidine,

at 37'

(GM-A)

(1.2

containing

At intervals,

0.1

filters

were

GAV).

The filters

points

a previously and washed

mg/ml)

at 27'

for

(100

for

first

to late

log

medium

(14).

60 set

was acid

and amino-

of the

"C-L-

to Millipore at 24-27'.

The cells

same medium

scintillation

from

medium

transport

Ug/ml)

of the

liquid

determined the

acid

by addition

1 ml portions

to vials

(mid

scale

30 min in an amino

(GM-GAV)

with

Large

Amino

was initiated

during

and mutant

once in growth

(GM-GAV).

chloramphenicol

were

acid

described

1.0 ml of medium

rates

the paren-

and methionine.

fatty

transferred

removed

obtained

arginine

were

twice

uptake

K12 JC411,

13 to 15 hours

ml aliquots

with

washed were

Initial

in

and vitamins

The reaction

on the

for

by centrifugation

cells

of the mutant

phosphatidylethanolamine.

weight))

acids

coli

Cl7 cyclopropane

including

(dry

urn) covered

experimental

leucine,

(2nJul).

(0.45

counting.

Escherichia

shaking

by preincubating

growth

amino

with

characterization

(14).

cellular

lipids

collected

glucose,

measured

for

excess

excess

to 0.8

and partial

elsewhere

auxotrophic

were

phase,

free

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

curves

(GM-

isotope drawn

through

6-8

of incubation.

Results Typical glutamic

acid,

strains port

JC411, activity.

system

nificant

acid change

4).

or E-52L previously

uptake

L-proline E-20

and E-52L.

These

the

Smaller

the

amounts were

uptake

changes

by performing

Both changes

and proline in

curves

respectively

and L-aspartic

However,

studied.

of glutamic

(Fig.

time-course

were uptake

used to culture

acid mutants

taken

of the

267

(and

acid

(or

changes

on the

amino

lysine),

There

glutamine)

other

presence

strains.

l-4

for

in transacid

and larger

uptake amounts

was no sig-

in either

in JC411 or reversed in the

L-

in Figs.

similar

up by the mutants.

produced

experiments

each

depending

of asparagine

L-asparagine,

illustrated

display

differ

of aspartic not

are

for

mutant

in strains

of spent The transport

medium changes

E-20

Vol. 50, No. 2, 1973

2 E 3

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

.

ll.l O4 2

JC

5 =q ii

411

7

? ; I

02

0

10

20 TIME

Fig. Figs. l-4: Ti%-co;r;;Lof 9-

L-Asparagine

Fig.

2.

L-Glutamic

might

metabolism

affected

of these

I 31

2.

JC411;

A , E-20;

acid

not be related

have

0 E coli 0.0!3 $i.-

I 20 (min.)

Fig.

Cl'-amino acid uptake by Amino acid concentration,

1.

which

TIME

1.

*

could

I IO

0

(mm)

Fig.

observed

OLJ

30

amino

to changes

the exchange acids

in the

endogenous

component

amino

of uptake.

was not significantly

acid

pools

Furthermore,

altered

in the

the

two

mutants. Increased asparagine sets

of data

more than lytic

were

transport observed

yielded

one uptake

components

for

rates over

hyperbolic mode for glutamate

for

glutamic

a broad

and decreased

concentration

reciprocal each

acid

amino

plots acid.

in JC411 having

26%

range

(Figs.

indicating

the

There the

appear

following

rates

for

5, 6).

Both

involvement

to be two catakinetic

constants:

of

Vol. 50,No.2,

BIOCHEMICAL

1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

06-

I 20

I 10 TIME hn)

Fig.

Kl,

0.003;

In both

mutants

L-Proline

Fig,

4.

L-Aspartic

V,,

the

uptake

in Table

I.

6.0;

acid

V2, 550 nmoles

Vmax of the higher

min-1

affinity

of the asparagine

rates Clearly

to the structural

tional

change

which

results

with

leucine

was necessary

relatively

3.

4.

data

100 mg-l

(cf.

15, 16).

component

(VT)

appeared

has not yet yielded

a similar

conclusion.

differently

it

Fig.

Analysis

Relative marized

ti;

only

to be elevated. clear-cut

Fig.

3.

Kp, 0.50

1 x

TIME (mm)

small

leads

for

unrepressed

representative

individual

amino

or metabolic

these

were amino

transport

acid

acids

in

component

269

with

by these variable,

the growth was measured.

acids

are sum-

catalysts

associated

of lipids particularly

amino transport

changes

to overproduction

and arginine

to include

several

the muta-

strains. possibly

medium

respond

The because

and only

a

Vol.50,No.2,

BIOCHEMICAL

1973

L-GLUTAMATE

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(mM)

L- ASPARAGINE

Pig. 6.

Fig. 5. Fig. Fig.

extracellular

acid of T4 C-L-glutamic glutamate concentration.

Initial rate extracellular

transport of T4 C-L-asparagine asparagine concentration.

Initial

5. 6.

(mM)

rate

transport as a function Symbols as in Fig.

of 1.

as a function of Symbols as in Fig. 1.

Discussion Besides

the defined

elsewhere

(14),

a number

of other

is

not yet

the

transport

other

hand,

changes

including

therefore,

a given

one or several aspect

of the

changes

other

composition

mutants alteration

in

lipid

findings

cell

, since

270

the

It

in amino

or metabolism.

On the

activity

are clearly catalysts

We view either

display

sensitivity.

by the transport

suggests

described

study

alterations

in transport

parameters. it

in this

in antibiotic

content

responses

altered

and excretion

utilized

relate

the changes

individual

of the

lipid

to causally

strain

indicating

in

overproducing

to specific for

heterogeneous

important

lipid

possible,

acid

changes

that

this

to

as the most

the membrane

Vol.50,No.2,

BIOCHEMICAL

1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I

RELATIVE

RATES OF AMINO ACID TRANSPORT IN E, -- COLI

PARENT AND LIPID

OVERPRODUCING MUTANTS

Transport Amino Acid*

JC411 nmoles

Asparagine

Rate Ratios

Rates

E52L E20 min-1 108 mg-1

E52L/JC411

E20/JC411

95

22

26

0.23

0.27

891

412

125

0.46

0.14

82

148

394

1.8

4.8

54

197

427

3.6

7.9

210

232

205

1.1

0.98

803

872

743

1.1

0.93

Arginine

69

76

113

1.1

1.6

Leucine

158

252

346

1.6

2.2

Lysine Glutamic

Acid

Proline Glutamine Aspartic

*All

Acid

amino

contains formly

acids

heterogeneous expressed

including

in surface

have been

confined

consequently,

this

thoroughly

explored.

and proline

auxotroph

grown

explanation immediate

this

aspect

of this vicinity

or that

proteins

Earlier

Esfahani

--et al.

profiles

different observation

for

differ

with

fatty that

of the relevant

(10)

recently

have

membrane cells

acids. lipid

acid

proteins,

and,

has not yet shown,

been

however, acid

of an unsaturated

molecules

auxotrophs

systems,

succinic

They propose

catalytic

271

bound

from

distributed

transport alteration

the

different

fatty

structure

uni-

catalysts

ways to uniformly studies

in

are not

the transport

to one or two saccharide of membrane

changes

are sufficiently

different

structure. largely

the mutational

structure,

in markedly

transport with

and that

such as binding

temperature-activity

genase

the

throughout

to respond

alteration

mM

regions

components

one another

the

at 0.03

dehydro-

fatty

as the

acid

most likely

may be dissimilar i.e.

that

that

in

membrane

Vol.50,No.

lipids

2, 1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are heterogeneously

different

proteins

molecules, immediate activities

distributed.

might

interact

An alternative

to different

extents

Thus even though the statistical vicinity might

protein-lipid

of these proteins vary differently

hydrocarbon

with

distribution

might be closely

interactions

is that

neighboring of lipids

similar,

as the temperature

chain

interpretation

lipid

in the

their

catalytic

was changed and the

were concomitantly

modified.

Acknowledgements This work Public

Health

was supported

Service

Grants

by National

Science

Foundation

Grant GB 23797,

CA-11186 and AM-13492 and by a fund established

in the name of the Marvin Hurley

Research

Fellowship.

REFERENCES 1.

Fox, C. F., Proc.

2.

Schairer,

3.

Wilson, G., Rose, S. P. and Fox, C. F., Biochem. Biophys. 2, 617 (1970).

4.

Overath, P., Schairer, 67, 606 (1970).

5.

Hsu, C. C. and Fox, C. F.,

6.

Wilson,

7.

Mindich,

8.

Willecke,

9.

Overath, P., Hill, 264 (1971).

H. U.,

Nat. Acad. Sci. and Overath,

U.S. 63, 850 (1969).

P., J. Mol. Biol.

H. U. and Stoffel,

W., Proc.

J. Bacterial.

Proc.

Nat. Acad. Sci.

K. and Mindich,

Res. Commun.

Nat. Acad. Sci.

55-, g(l971).

U.S. 68, 420 (1971).

L., J. Bacterial.

9,

F. F. and Lamnek-Hirsch,

I.,

514 (1971). Nature

New Biol.

10.

A. R., Knutton, S., Oka, T. and Wakil, Esfahani, M., Limbrick, Proc. Nat. Acad. Sci. U.S. 68, 3180 (1971).

11.

Holden,

12.

Holden, J. T., Hild, O., Wong-Leung, Y. L. and Rouser, Biophys. Res. Cotmnun. 40-, 123 (1972).

13.

Holden, (1972).

14.

Kenyon, C. N. and Hegeman, G. D.

15.

Reid, K. G., Utech, (1970).

16.

Halpern,

3. T. and Utech,

N. M., Biochim.

Biophys.

J. T. and Bunch, J. M., Biochem. Biophys. Manuscript

N. M. and Holden,

Y. S. and Even-Shoshan,

U.S.

103, 410 (1970).

G. and Fox, C. F., J. Mol. Biol. L.,

44, 209 (1969).

S. J.,

Acta 135, 517 (1967). G., Biochem.

Res. Commun. 46-, 437

in preparation.

J. T., J. Biol.

A., J. Bacterial. 272

234,

Chem. 245, 5261

93, 1009 (1967).