Stimulation of the Ca2+-dependent polymerization of synexin by cis -unsaturated fatty acids

Vol.

132,

October

No. 2, 1985 30,

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS Pages

1985

STIMULATION OF THE Ca*+-DEPENDENT OF SYNEXIN BY =-UNSATURATED DAVID

C.

STERNER,

De,partment

of

Neuroscience,

Received

August

19,

WILLIAM

J.

Pharmacology and Cell University Charlottesville,

505-512

POLYMERIZATION FATTY ACIDS

ZAKS

AND CARL

and Programs and Molecular of Virginia VA 22908

in

E.

CREUTZ

Biophysics, Biology

1985

The influence of fatty acids on the polymerization of synexin was studied by monitoring light scattered from solutions of purified synexin. m-unsaturated fatty acids such as arachidonate or oleate stimulated synexin polymerization at sub-micromolar concentrations, while saturated fatty acids, a trans unsaturated fatty acid or a fatty acid methyl ester had little effect. The olymerization of synexin occurred at lower concentrations of Ca 4+ in the presence of the fatty acids than Therefore, Ca*+ and free fatty in the absence of fatty acids. acids may act as co-regulators of synexin action in stimulated 0 1985 Academic Press, Inc. secretory cells.

In

an

membrane

attempt

have in

have'been

identified

promote membrane

undertaken

the

both

that

close

These

the

case

of

mechanism

by which

follows:

1)

with

Kd -4

Ca2+

then

M (threshold

irreversible

When

uM, binds

to

- 10 membranes contact

to

one

synexin

a site

binds

to

membranes

polymerize form

between

one

to

proteins but

also

another, and

hypothesis

on

bind

two

membrane

to

causes

that

(1)

specific

of

several

least

to

promotes

class

regulate

membranes,

membranes

binds

PM) which

to

At

bind

are

protein

a second

to

(l-5).

proteins

this

synexin

proteins

of

synexin,

Ca2+

soluble

only

attachment

might

exocytosis,

Ca*+

not

that

during of

of

presence

fusion. In

proteins

occurring

been

membranes

different

identify

interactions

studies

(5).

to

and

calelectrin for

the

contacts

is

as

the

synexin

molecule

as

a monomer

(6);

site

Kd - 200

binding

the

synexin

(71,

thus

with

monomers promoting

the

membranes.

A2

Copyright 0 1985 rights of reproducfion

2)

on an Once

the

0006-291X/85 505

u

$1.50

by Academic Press, Inc. in any form reserved.

Vol.

132,

No. 2, 1985

membranes low

BIOCHEMICAL

have

concentrations

might

become

result

of

of

the

from

properties action The like

most

synexin

secretory

cells

necessary

to

may

mediate is

the

we have in

sensitivity

it

of

communication

the

synexin

sought

the we

cells

synexin describe

between

synexin

and

synexin

polymerization,

to

free

the

as a

Interestingly,

same

fusion

quite

mechanism

that

that

in

Ca2+

of

that

if

could

a protein

stimulated appears

polymerization

determine

to

other alter

important that at

appears low

be

reaction. cofactors the

may

Ca2+

reaction.

a potentially

particularly

cell

hypothesis

of

acids

as

polymerization

the

polymerization

fatty

to

(9).

membrane level

stimulated

and

contacts

high

exposed

immunochemically

possess

of

if acids,

(8).

binding

aspect

stimulate

Accordingly, present

Ca2+

membrane

might

fuse

secretory

and

has

COMMUNICATIONS

fatty

phospholipases

physically

troubling

will

a stimulated of

suggest

promoting

they

RESEARCH

a-unsaturated

in

synexin,

that in

free,

is

BIOPHYSICAL

contact,

activation which

distinct

this

available

calelectrin,

be

formed

AND

In

this

interaction to

concentrations

stimulate of

Ca2+.

MATERIALS

AND METHODS

Materials. Synexin was prepared from bovine liver by ammonium sulfate precipitation from a post microsomal supernatant and gel filtration on LKB Ultrogel ACA 34 (1). The synexin was isolated in 0.3 M sucrose, 40 mM MES pH 6.0; immediately before the light 40 mM HEPES was added and the pH scattering experiments, Fatty acids were obtained from Sigma adjusted to 7.3 with NaOH. and stock solutions of 5 mg/ml were prepared in ethanol. Calcium buffers were prepared as 50X stock solutions using CaC12 and EGTA according to the method described by Caldwell (10). In calculating the pCa, a binding constant of 1.897 x lo7 for Ca2+ and EGTA at pH 7.3 was used (10). Light scattering measurements. Intensity of 350 nm light scattered at 90’ was monitored continuously in a Spex 1llC spectroflurometer using a 0.5 cm square cuvette. The following additions were made to the cuvette: 263 ul synexin in 0.3 M surcose, 40 mM MES, 40 mM HEPES, pH 7.3; 66 nl 0.15 M kC1; 4.5 ~1 ethanol containing various concentrations of fatty acid (also included in controls without fatty acid). After recording baseline light scattering for 200 set, synexin polymerization was initiated by adding 6 ~1 506

Vol.

132,

No.

of Ca2+ various monitored

BIOCHEMICAL

2. 1985

AND

BIOPHYSICAL

RESEARCH

buffer to give a final EGTA concentration amounts of CaC12. Polymerization was for 400 sec.

COMMUNtCATtONS

of 2.5 subsequently

mM and

RESULTS The by

polymerization

measurement

of

of

light

spectroflourometer line

for

light

form,

of

Polymerization

scattering

If

reaction added no

2.2

mixture, is

greatly

increase

in

0.75E

the

(7)

and

PM arachidonic

acid

by

scattering

is

stable

in

upon

addition

level

in

2 to

data

(11)

these

included

no

the

the light

under

If seen

a base

the

response

1).

a standard

synexin

and

is

scattering (Fig.

in

centrifugution

occurs

increased light

a new

monitored

establishing

immediately to

90°

initiated

polymerization

light

at

from is

microscopy

conveniently

After

intensity

begins

that

conditions.

1).

increases

Electron confirmed

(Figure

be

scattering

polymerization

intensity

minutes. have

the

can

nm)

scattering

monomeric Ca2+.

(350

cell

the

synexin

in when

protein addition

the

Ca2+ is

is

present, of

Ca2+

05 300.00

Position Figure

1:

(secl

Intensity of light scattered from synexin solutions undergoing Ca*+ dependent polymerization. Raw data from fluorometer is plotted as intensity in photons per second versus time ("position") in seconds. At 200 seconds polymerization was initiated by adding 400 IJM Ca2+. Traces 3,4: 1 IJM synexin; traces 1,2: 1 VI4 synexin, 2.2 LIM arachidonic acid; traces 5,6: 2.2 PM arachidonic acid without synexin. 507

3

Vol.

132,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-

125

-5

Figure

2:

Stimulation of Ca2+ dependent synexin polymerization as a function of fatty acid concentration. Polymerization of 1 PM synexin initiated by the addition of 400 UM Ca2+ in the presence of various concentrations of oleic acid (solid line), arachidonic acid (dashed line) or myristic acid (dotted line). Percent stimulation = If-a -Io x 100, 10

where If a = increase in intensity of light scattered by a synexin solution containing added fatty acid, 400 set after the initiation of polymerization by the addition of Ca2+; and 10 = increase in intensity seen with synexin alone. The data points represent the mean + range for duplicate experiments.

(Fig.

indicating

11,

cation

and

the

scattering

fatty

stimulation

concentrations

of

molecules

(Fig.

interaction

not

between

responsible

synexin acid, of

Th is

for

with

small

Table

stimulating

The

one

methyl

Elaidic

necessary acid,

ineffective of

to

the

trans

(Table

1).

the

fatty

fatty

the

the

light

occurs

at

be

an

fatty

acids

ester

of

the

isomer It acid

is that

per

that

perhaps

The

greatest

stimulation

introduced acid

that

oleic

striking are 508

critical

acid

(Table

that

was the for

was

ineffective

a free

acid,

fa ttY

(Fig.

was

interaction

of

the

synexin,

oleic

at

seen

synexin

of

were

low

being

four

indication

suggesting promote

effects

acid

complex.

polymerization, is

initial

oligomers

a-unsaturated 1).

polymerization w ith

may

a nucleation

when

features

fatty

2).

interacts

group

is

ratios

stabilizing

and

acid

of

substoichiometic

seen

a direct

increase.

The

acid

that

1). also

structural promoting

head

2 at

Vol.

132,

No. 2, 1985

BIOCHEMICAL

Table

I.

AND

Stimulation

of by

Fatty

BIOPHYSICAL

RESEARCH

synexin acids

fatty

polymerization

acida

% stimulationb 0 +

IlOne

01 eic methyl

oleic

synexin the

Mean

The synexin (Fig. at

of

two

presence

+

1

of fatty PM synexin. =

range

If.a

acid,

- Io I0

lower

concentrations

Figure

3:

3

free

fatty at

the

all

of

100 in

without

fatty

acid

those

was

required

contact to of

stimulation 90%

at

to by

found

concentrations

Ca2+:

acid.

determinations.

into

percentage

16

rig/ml

increase

to

brought

5

0.65 x

duplicate

similar

membranes of

However,

for

are

polymerization 3).

+

16

polymerization

fusion

20

529

Where If a = intensity presence’of fatty acid. IO = intensity increase C.

2

palmitic

% stimulation

b.

+

321

Concentration pCa 3.4,

3c

96

myristic

elaidic a.

COMMUNICATIONS

nM

synexin

(8).

stimulate Ca2+

was 100

promote

Ca2+;

tested the

greatest 158%

at

Ca2+ dependence of synexin (1 PM) polymerization in the absence (solid line) or presence (dashed line) The intensity scale has of oleic acid (2.3 PM). been normalized so that 100 = increase in light scattered by 1 UM synexin alone 400 set after the The data represent the addition of 400 uM Ca2+. mean + standard deviation for the means from five independent experiments each conducted in duplicate. 509

10 1~

Vol.

132,

No.

2, 1985

M Ca2+;

and

curve

shows

as well is

BIOCHEMICAL

as

490%

at

4 UM Ca2+.

both

an

increase

a shift

reduced

AND

from

to 200

In in

the

the

maximum

so

35

RESEARCH

effect,

the

left

pM to

BIOPHYSICAL

that

COMMUNICATIONS

Ca2+

titration

polymer

the

formation,

apparent

Kd for

Ca2+

PM.

DISCUSSION The

apparent

shift

the

presence

with

the

behavior

of

Ca2’

and

lipids.

For

Ca2+

is

increased

when

this

protein

reported

of

free

is

the

fatty case

of

action sensitivity yet

of

to

be

interact interaction The stimulated

these

two

Ca2+

that

of

of

critical

interaction synexin-dependent presence

of

polymerization

interaction

between

provide chromaf

Ca2+:

When

f in

chromaffin

to

been of

Ca2+

interesting

diacyl

glycerol

of

or

the

Ca2’ general,

proteins

as that

allows

to

occur

in

secretion less

of

fatty

than

free

on

or acids

from 10

fatty off

liM. acid

(Fig. and

a curious

e membrane

fusion are

At may

3).

synexin

Furthermore, for

granules 510

bound

most

that

promote

explanation granul

are

sites.

importance. the

for

some

generally

synexin

albumin

be

expected

availability

serum

in

binding

that

both

a shift

cations

be

with

phospholipase

structure

of

might

physiological may

of

may

the

types

are

the

either

divalent

or

models, Ca2’,

this

and

cells,

cell

Therefore,

of

interact

C has

products

It

analogy

acids

Perhaps

where

in

concentrations

cause

(14).

the

turn

C,

feature

secretory

essentially

be

enzyme

of

low

(13).

phospholipids,

between

of

at

both

lipids

levels

affinity fatty

present

both

permeabilized

that

synexin

interesting

phospholipase

acids,

the

levels

proteins the

kinase

recognized, with

an

activity

fatty

of

provides

addition,

are

on membrane

sensitivity

&-unsaturated

protein

cis-unsaturated

other

example,

greater

acids

acid

some

In

have

Ca 2+

the

fatty

(12). to

if

free

in

may

this feature in

aggregated

of

the by

Vol.

132,

No. 2, 1985

synexin

and

BIOCHEMICAL

then

chemiosmotic fuse,

that

are

not

are

specifically

the

then

possible

synexin the

contact

membrane

structure.

where

study

that

it

lipids

affinity

will

bind

that

it

of fatty a

will

be

competing essential

to

membrane

fusion

is

to

the

dependence

presence

been

used

used

to

of to

answer

unpredictable the will

application be

Ca 2’ fatty

acids

study

membrane

this

question

manner

by

of

sucessful

more in

The

membrane

it fusion

answering

This

study Foundation

was

supported (PCM 8206453).

acid.

if

the

synexin

by

511

enough membrane In

Ca2+ and

fatty

in

assay

synexin

cannot

affected

in It

that

is

hoped approaches

questions.

grant

from

the

has be

an

Em_T a

acids

polymerization

biophysical these

areas

high

fatty

(8).

sophisticated

ACKNOWLEDG

Science

is

on

is

of

by

since

of

determine

turbidity

aggregation

acid,

to

presence

synexin

alone.

fatty

additional

determine by

location

effect

acid

for

fatty

regions

important

the

of

the

important

induced

the

disruptive

“sink”

of

the

in

the

be

these

fatty

even

dependence similar

be for

acid

may

only in

does

binds

two

will

synexin

resulting

in

to

it

break

this

a

leave

membranes

membrane-membrane

have

regions

a-unsaturated

of

synexin

at

if

why

if

do

and

arises,

that

could

break

another,

is

leads

provide it

the

concentrated

First,

the

addition,

be

therefore

investigation.

whether

and, may

system,

region

answer

acid

membrane

of

the

granules

contacts

however,

question

molecules

fatty

Our

The

the

simply

one

COMMUNICATIONS

activating

ATP,

(15);

with

weaken One

of

membrane

this

contact

(8).

by

membranes

forming

to

of

fusion

contact? of

added

RESEARCH

stress

presence

junctions

regions

membrane

the

in

BIOPHYSICAL

osmotic

granule

membrane

acids their

the

involved

firm

acid

in

instead

behind

at

to

swelling

not

fatty

exposed

AND

National

that

Vol. 132, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Creutz, C.E., Pazoles, C.J. and Pollard, H.B. (1978) J. Biol. Chem. 253, 2858-2866. Creutz, C.E., Dowling, L.G., Sando, J.J., Villar-Palasi, C Whipple, J.H. and Zaks, W.J. (1983) J. Biol. Chem. 258, 11;64-14674. Geisow, M.J. and Burgoyne, R.D. (1982) J. Neurochem. 38, 1735-1741. Bader, M.F., Hikita, T. and Trifaro, J.M. (1985) J. Neurochem. 44, 526-539. Sudhof, T.C., Ebbecke, M., Walker, J.H., Fristsche, U. and Boustead, C. (1984) Biochemistry 23, 1103-1109. Creutz, C.E. and Sterner, D.C. (1983) Biochem. Biophys. Comm. 114, 355-364. Res. Creutz, C.E., Pazoles, C.J. and Pollard, H.B. (1979) J. Biol. Chem. 254, 553-558. (1981) J. Cell. Biol. 9l, 247-256. Creutz, C.E. Sudhof, T.C., Walker, J.H. and Obrocki, J. (1982) The EMBO Journal, 1, 1167-1170. Caldwell, P.C. (1970) in Calcium and Cellular Function (Cuthbert, A.W., ed.) pp. 10-16, McMillan, London. Morris, S.J., Hughes, J.M.X. and Whittaker, V.P. (1982) J. Neurochem. 39, 529-536. Aguanno, J.J. and Ladenson, J.H. (1982) J. Biol. Chem. 257, 8745-8748. Takenawa, T. and Yoshitaka N. (1981) J. Biol. Chem. 256, 6769-6775. McPhail, L.C., Clayton, C.C. and Snyderman, R. (1984) Science 224, 622-625. Creutz, C.E., Zaks, W.J., Hamman, H.C. and Martin, W.H. in Cell Fusion (A.E. Sowers, editor) Plenum Publishing Corp. 1986. -in press

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