Phosphorylation of a sugar-specific protein component of the lactose transport system in Staphylococcus aureus

Vol. 42, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PHOSPHORYLATION OF A SUGAR-SPECIFIC

PROTEIN COMPONENT OF THE LACTOSE

TRANSPORT SYSTEM IN STAPHYLOCOCCUS AUREUS* Teruko

Nakazawa,

Robert

D. Simoni,

John B. Hays+ and Saul

Roseman

McCollum-Pratt Institute and the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

Received January 14, 1971 Summary A sugar-specific co onent of the lactose transport system inStaphyZoas an intermediate in the auzws, Factor III mPac, is phosphorylated over-all transfer of a phosphoryl group from PEP to lactose. p-mlac is isolated and shown to be a substrate for the final phosphoryl transfer reaction to sugar, catalyzed by Enzyme II1ac.

coccus

Many bacterial mediates

species

the transport

(1) 0 The reaction

this

over-all

found

weights

to sugar.

Initial

reaction

resulted

from

I]

[Enzyme

II]

to 10,000.

As will

phospho-Enzyme The transfer

*

Supported American Cancer tute. t Postdoctoral

PEP + HPrP-HPr

studies at least

the

cell

of phosphate (2) with

two distinct

Sugar-P

sugars

fraction three

be reported

(PTS) which membrane from

the PTS showed steps:

P-HPr + Pyruvate

common to all

from

across

the transfer

+ Sugar---t

in the soluble

of the HPr proteins

9,000 I is

primarily

by the PTS is

[Enzyme

system

of carbohydrates

(PEP)

Enzyme I and HPr are are

a phosphotransferase

of a variety catalyzed

phosphoenolpyruvate that

contain

elsewhere,

+ HPr

phosphorylated

of cell

different

(1)

by the PTS and

lysates.

organisms

(2)

The molecular are in

the intermediate

the range in Reaction

I. of the phosphoryl

by Grant Society. Fellow

group

AM-9851 from Contribution

from P-HPr

to sugar

requires

the USPHS and Grant P-544 from No. 608 from the McCollum-Pratt

of the American

836

Cancer

Society.

the

the Insti-

of

Vol. 42, No. 5, 1971

sugar-specific

components

such systems at least tive

far

studies

all

via

sugars

(Factor

with

In all

two proteins

For example,

the E.

two proteins,

S. aureus,

IIIlaC).

are

required, constitu-

COli

as well

series

apparently (5)

protein

The p resent group

which

we reported

of a membrane

of the phosphoryl

the following

laboratory,

into

the PTS (4),

the induction

protein

dissociated

are inducib1e.I

as lipid

and

(3).

In earlier

quires

in this

is membrane-bound.

has been

cation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the PTS, most of which

analyzed

one of which

divalent

fer

thus

Enzyme II

izes

BIOCHEMICAL

lac

provide

to lactose

re-

> and a cytoplasmic

evidence

(and

and util-

utilization

lactose

(Enzyme II

studies

from P-RPr

that

transports

its

that

the

analogues)

trans-

involves

of reactions: 2P-HPr

+ IIIlaC

[Enzyme I11aC]P2-IIIlaC

c---, P2-IIIlaC

+ 2 lactose

-

+ 2RPr

(3)

2 lactose-P

lac

+ III

(4)

EXPERIMENTAL S. aureua

5601

cells

were

bactopeptone

which

scale

by Dr.

Thomas Stoudt

these

experiments

prepared ials

with

induces

would

according

were

extracts

glass

beads

were

Enzyme II

lac was purified followed

commercial prepared

in an Eppenbach

elsewhere

cholate

of Merck

system and Co.,

containing (5).

in detail,

by 0.005

previously

and 1x

were grown

on large

whose

generous

32 P-enolpyruvate reported

(3).

assistance was

All

other

mater-

sources. from

0.5

to 1 kg of cells

Micro-mill.

The purification

separated

the PTS components

by extracting M NaOH.

1% galactose

Cells

without

not have been possible.

from

be reported

in media

the lactose

to the procedure

obtained

Crude

grown

washed

The crude

membranes supernatant

lIn accord with recommended nomenclature (3,5), IIlac and Factor IIIlac designate the sugar-specific the PTS obtained when cells are grown under inducing thio - B - 2 - galactopyranoside.

837

by homogenization procedures,

to

from each other.

with

1% sodium

fluid

containing

deoxy-

the abbreviations Enzyme (lactose) components of conditions. TMG = methyl

Vol. 42, No. 5, 1971

to pH 4.9;

DEAE-cellulose followed

HPr remained

llllac

lac

phosphate)

followed

The following

about

and contained

peared

was analyzed trifugation 9,000

residue

a molecular

and 12,000,

37,000.

it

composed

about

about

200-fold; (disc-

methods

for

IIIlaC

de-

ap-

ultracentrifuga-

However,

electrophoresis,

is

while

M potassium

Factor

weight

that

M KCl),

three

9,000.

weight

Enzyme I

homogeneous

and equilibrium

sulfate-gel

(40 to 80%);

ultracentrifugation),

per mole of protein;

the molecular

indicating

HPr,

electrophoresis

dodecyl

in 4 M guanidine,

20-fold; essentially

of about

The

lac Enzyme II

attained:

of about

(0 to 0.2

M KCl).

and equilibrium

gave values

(disc-gel

by sodium

and 4.5

to 0.35 (0.15

to 0.20

The HPr appeared

weight

and showed

(0.2

Enzyme I about

(65 to 100%)

chromatography.

(0.12

membranes;

by

phosphate).

G-75

by DEAE-cellulose

were

and was purified

ammonium sulfate

on hydroxylapatite

1 histidine

molecular

with

by chromatography

factors

ad-

DEAK-cellulose

M potassium

by Sephadex

and subsequently

precipitation

on DEAK-cellulose

at pH 9.5

homogeneous

tion)

to 0.02

separated

400-fold.

electrophoresis

termining

were

fluid

G-75,

and treated

purification

over washed

and IIIlaC gel

(0.001

by chromatography

was isolated

lo-fold

on Sephadex

was dissolved

Enzyme I and III

streptomycin

the supematant

chromatography

pH 4.9 precipitate

with

and ammonium sulfate

and hydroxylapatite

was purified

in

chromatography

by column

M KCl),

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

III lac , and HPr was treated

Enzyme I, justed

BIOCHEMICAL

when

III lac

and by ultracen-

appeared

to be between

of 3 to 4 subunits.

RESULTS Transfer

of the Phosphoryl

Previous reaction

work

IIIlaC

Enzyme I andHPr. mation

established

was subsequent

show that

(data

incorporated

not per

Group

that

to P-HPr

shown).

of either

of III lac

participation

formation.

was phosphorylated Omission

III lac

from PEP to Factor

in

The data

the presence

the over-all

presented

in Table

I

of catalytic

amounts

of

I or HPr resulted

It

in

in no P-III

lac

for-

is also apparent that two phosphoryl groups were lac lac mole of III . 32P*-III has been isolated from large

838

Vol. 42, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

Table

Transfer

I

of Phosphate from 32PEPto Factor IIIZaC

Incubations

Time

32P-III

Ratio

min.

cpm

of 32P to IIIlaC

1. 111lac

+ 32 PEP (1 mumole)

15

8,800

1.64

2. llllac:

+ 32 PEP (1 mumole)

30

7,950

1.52

3. IIIlac

+ 32 PEP (10 mnmoles)

10

10,500

1.94

4. llllac.

+ 32 PEP (10 mnmoles)

20

9,750

1.88

5.

+ 32PEP (1 mpmole)

30

-

260

-

Incubations were conducted at 37" and contained III la' (10 ug, 0.27 mnmole); Enzyme I, 8.2 ug for incubations 1, 2, and 5, and 16.4 ug for 3 and 4; HPr, 0.2 u ; potassium phosphate buffer, pH 8.3, 0.5 umole; MgC12, 50 mumoles; and 3f PEP, 1.94 x lo7 cpmfpmole as indicated. The total volumes of incubations 1, 2, and 5 were 35 1.11 and 3 and 4, 55 ~1. EDTA (1 umole) was added to stop the reaction. Aliquots of each incubation were spotted on strips of Whatman DE 81 (DRAK) paper (2 x 10 cm) and subjected to ascending chromatography in 30% ethanol-O.05 M KCl-1 mM EDTA-10 mM Tris-HCl buffer, pH 8. Under these conditions 32P-IIIlac remained at the origin and 32PEP and 32Pi migrate near the solvent front. The chromatograms were then cut into 1 cm strips and counted in toluene liquid scintillation fluid.

scale

incubations

the phosphoryl

linkage

to that

was similar linkage

by chromatography examined. obtained

lac

in P-III

on Sephadex

is

At pH 3.6

for

and 46"

the rate

from E. coZi,

P-HPr

to a histidine

G-75 and the pH stability

residue

thus

of hydrolysis

suggesting

(as has been

of

that

demonstrated

the for

P-HPr). Transfer

of the Phosphoryl

The direct demonstrated

transfer in Figure

G-75 and was free then

incubated

under

by separating

protein

preparations

required

inhibition

for

from P-HPr

of the phosphoryl 1.

32P-HPr

the

conditions

the were

the transfer.

by EDTA),

group

3)

to III lac

is

by chromatography on Sephadex 32 proteins. P-HPr and IIIlaC were 1 and 32P transfer

by G-75 chromatography.

we assume that The reaction

and phosphoryl

(Reaction

from P-HPr

shown in Figure

two proteins pure,

to III la'

was isolated

of 32PEP and extraneous

assayed

is

Group

transfer

839

no additional

showed

no metal

was very

rapid:

Since protein requirement incubation

was both component (no at 0"

Vol. 42, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

600

A C 2 t

a

a N m

600

0.75

200

0.25 40

60

80

0) .z -0 $

100

Fraction Number Fig. 1. Transfer of the J$osphate from 32P-HPr to IIIZaC. In5ybation P-HPr mixtures were as follows: PHPr, 0.6 mpmole, 42,000 cpm (this preparation also contained 0.54 mpmole of HPr since it was not completely phosphorylated in these experiments); MgC12,lnmole; in the absence (A) or presence (B) of 4.15 mpmoles of IIIlac in a total volume of 500 pl. Incubation was carried out at 37" for 2 min, 500 ~1 of water were added and the total reaction mixture was applied to a Sephadex G-75 column (1.8 x 40 cm) which was equilibrated with 1 mM Tris-Cl buffer, 50 mM KCl, pH 9. Fractions were collected and aliquots removed for 32P determination (O), enzymatic assay of HPr (0) and IIIlac (0). In addition results obtained by 1) substituting 10 pmoles of EDTA for MgC12 or 2) incubation for 0 min at 0' instead of for 2 min at 37", were the same as those shown in B. The enzymatic assays for HPr and IIIlaC were done by measuring 14C-thiomethyl galactoside-P formation as previously reported (5). The small 32P peak at the end of the chromatogram is 32Pi which was present in the 32P-HPr'preparation. All samples were counted in Triton-toluene liquid scintillation solution. followed

by rapid

bation

at 37".

phoryl

group

separation The reaction

an accurate

liminary

results

32P-HPr

subunits ally

of this

acetylated

the labeled

shown

constant is

in Figure

bound

protein.

has not

HPr was phosphorylated

yet been

lac

anhydride (0.8

that

840

of the phos(data

not

shown).

determined,

pre-

32P was transferred would

or if

it

these

possibilities,

(2 moles mole

as incu-

1.

1 indicate

To eliminate

14C-acetic

demonstrable

the same results

to III

same results

and transfer

approximately

essentially

tightly

with

it

the

reversible

to HPr was easily

show that

to IIIlaC, were

freely

equilibrium

the results

from 32P-HPr if

is

from =p-IIIlaC

Although

While

at 4" gave essentially

exchanged

have been with

one of the

HPr was chemic-

of 14C per mole

of 32P per mole

obtained

of HPr),

of I&C-HPr),

and

Vol. 42, No. 5, 1971

the doubly Virtually

BIOCHEMICAL

labeled

product

complete

transfer

in the RPr peak.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was incubated

III lac

with

of 32P was obtained,

Thus we conclude

that

as described

while

Reaction

all

3 (see

in Figure

1.

of the 14C remained

Introduction)

is

es-

tablished. Transfer

of the Phosphoryl

In order indeed

an active

lated

that

intermediate

by chromatography

similar with

to ensure

to that

IIlaC

(Table

in

in Table

lac

to 'JMG (Reaction

formed

in previous

the over-all

I.

and TMG; TMG-32P

of P-III

The data for

the P-III

lac

reaction, a large-scale

The isolated

32P-II11aC

formed

reactions

32P-II11aC

G-75 from

was only

4)

in

incubation was then

the presence

was

was isomixture incubated of Enzyme

II).

Interaction

specific

from P-III

on Sephadex

shown lac

Enzyme II

Group

lac

presented

above

lactose

Enzyme II lac

and TMG with

and its

and reported analogues,

Table Transfer

of phosphate

previously and that

it

(5) acts

show that

as an intermediate

II from

32P-IIIZaC to TMG 32P -111 Remaining

Incubation

1. Complete

32P-TMG Formed

cpm

cpm

634

3200

2. Minus

Enzyme II lac

3627

0

3. Minus

TMG

3428

0

3745

0

4. Minus

Enzyme II

lac

and TMG

The complete incubation contained dithiothreitol, pmole; Tris-HCl buffer, pH 9, 2.5 umoles; Enzyme IIlac, 33 pg; Mgc12, 0.05 nmole; 32P-IIIlac, 0.137 mpmole, 4800 cpm; in a total volume of 85 ~1. Incubation was conducted at 37' for 2 min. Aliquots of each incubation were spotted on Whatman No. 3MN paper and subjected to high voltage electrophoresis in 1% sodium Under these contetraborate for 20 min at 4000 volts. ditions 32P-III remains at the origin and 32P-TMG moves toward the anode. The paper strips were cut into 2 cm strips and counted in toluene scintillation fluid. 0.5

841

IIIlaC

is

Vol. 42, No. 5, 1971

phosphate

carrier

substrates

for

carried

BIOCHEMICAL

between

P-HPr

Enzyme II lac .

out in

an attempt and TMG);

sively

the results

system

Figure

2 shows

for

lac

P-III

was phosphorylated. tration

indicate

tween

studies

including

currently

transfer product

were

lac

for 0.03

this

caution.

lac

using

a generating

added

to the system

with

increasing

concenlac

Km at 0.0 M P-III

formation

and excludes

patterns

to define

exten-

and at

mM and 1.0 mM, respectively.

of P from P-III

inhibition

in progress

the III

TMG increased

involving

TMG, and Enzyme II

intermediate

all

values

anorderedmechanism

P-IIIlaC,

where Km for

lac

with

were

components

and was not

of TMG-P synthesis,

conditions

of P-III

is particulate

are

studies

of the three

be interpreted

rates

and sugar

kinetic

the relationship

should

under

Both P-IIIlaC

some preliminary

Enzyme II

lac ; the estimated

volving

are

since

the initial

concentration

data

Therefore,

The apparent

of P-III

infinite

and the sugar.

to clarify

lac , P-IIIlaC (11 purified,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lac

of a ternary a "ping-pang"

to Enzyme II lac .

and direct

the mechanism

binding

of the

last

The

complex

be-

mechanism

in-

Other measurements

reaction.

25 I/S

[ l/n&

m’lil-I

Fig. 2. Kinetics of the Enzyme II lac Reaction. Incubations contained potassium phosphate buffer, 20 pmoles, pH 7.5; phosphoetvl.Iyruvate, 2 umoles; KF, 0.5 pmole; dithiothreitol, 1 umole; Enzyme IIlac 33 pg; Enzyme I, 35 pg; HPr, 15 ug; MgC12, 1 pmole; IIIlac, 1, 2, 5 pg as indicated (the IIIlac used in these experiments was not completely pure and therefore no molar values are given); 14C-thiomethyl B-D-galactoside, concentrations as shown. Final volume was 240 ul. Incubation was at 37" for 30 min and the TMG-P was detected as previously reported (5). DISCUSSION The data

presented

in

this

paper

established 842

the

reaction

sequence

for

BIOCHEMICAL

Vol. 42, No. 5, 1971

the lactose llGac

phosphotransferase

represents

of an isolated IIIlaC

of :Lactose

that

P-IIIlaC

Since

IIIlaC

groups the

cific

solute-specific

transport

together and its

is

the

form

the

analogues

direct

remaining

subunit(s) of III

Enzyme II

lac

lac

this

apparently than

It

covalent is

carrier organism.

it

involved

in

results a direct

is

between

attractive

its

interaction

P-HPr

with

trans-

and II

lac .

2 phosphoryl

to speculate

interaction with

the

that

demonstrate

and only

the interactions from

alteration

for

The data

protein

of

our contention

complex

of 3 to 4 subunits,

per molecule, are

rather

protein.

energy-coupling

seems to be comprised

The phosphorylation

of a reversible

functional in

RESEARCH COMMUNICATIONS

in S. aureus.

demonstration

can be incorporated

specificity

system

the first

and IIlaC

port

AND BIOPHYSICAL

II with

lac .

that The

the spe-

sugar.

REFERENCES 1. Roseman, Saul, J. Gen. Physiol. 54, 138s (1969). 2. Kundig, W., Chosh, S., and Roseman, S., PPOC.Nat. Acad. Sci. U.S.A. 52, 1067 (1969). 3. Kundig, W., and Roseman, S., J. Bioi!. &em. 246, in press. 4. Egan, J.B. and Morse, M-L., Bioehim. Biophys. Acta 97, 310 (1965); 112, 63 (1966). 5. Simoni, R.D., Smith, M.F. and Roseman, S., Biochem. Biophys. Res. Common. 32, 804 (1968).

843