Secretory vesicle — Cytosol interactions in exocytosis: Isolation by Ca2+-dependent affinity chromatography of proteins that bind to the chromaffin granule membrane

Vol.

103,

Nd.

December

BIOCHEMICAL

4,198l

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1395-1400

31, 1981

SECRETORY VESICLE - CYTOSOL INTERACTIONS IN EXOCYTOSIS: ISOLATION BY Ca2+-DEPENDENT AFFINITY CHROMATOGRAPHY OF PROTEINS THAT BIND TO THE CHROMAFFIN GRANULE MEMBRANE. CARL E. CREUTZ Cell Biology and Biochemistry Section, Clinical Hematology Branch, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, Md., and Department of Pharmacologyl, University of Virginia, Charlottesville, Virginia 22908 Received

November

16.

1981 SUMMARY

Proteins from adrenal medullary cyipol that bind to chromaffin granule membranes in the presence of Ca were isolated by affinity chromatography on granule membranes coupled to Sepharose4B. C tosol was applied to the affinity column in the presence of 2 mM free Ca k . One group of proteins was eluted at 50 pM Ca2+ and had molecular wei hts of 60,000, 46,000, 36,000, 34,000, 32,000 and 26,000. At 0.1 pM Ca 2+ additional proteins of molecular weights 70,000, 44,000 and 33,000 were eluted. Both groups of proteins aggregated isolated chromaffin granules in the presence of Ca2+. Since exocytosis involves cytosol-membrane interactions regulated by Ca2+, these proteins may have functional roles in this process. The term "chromobindins" is introduced to describe these proteins. INTRODUCTION Catecholamines cell

by exocytosis.

granule) in

cytoplasm. secretion proteins plasmic

attaches

exocytosis,

appear

from

process to,

the membrane by the

Ca2+ may interact

directly

with

to orchestrate elements

that

the adrenal the

secretory

and fuses

to be regulated

and also

Present

released

In this

approaches,

compound

events

1.

are

of another

with structural,

the overall

process.

a part

in these

chromaffin

vesicle

(chromaffin

the plasma

membrane,

secretory

concentration

cytoplasmic

play

with

medullary

vesicle.

involved

contractile It events

in

or regulatory

seems likely must

These

Ca 2+ in the

of free

the membranes

or,

interact

that

cyto-

physically

address.

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Vol. 103, No. 4,1981

with

the secretory

regulated

by Ca2+,

be isolated cytosol

vesicle This

and that

consideration

granule

the feasibility

AND BIOPHYSICAL

membrane

by Ca2+-dependent

on chromaffin

demonstrate

BIOCHEMICAL

their

suggested

affinity

RESEARCH

interaction that

chromatography

membranes.

COMMUNICATIONS

The results

might

such factors of adrenal described

be might

medullary here

of such an approach.

METHODS Preparation of the Affinity Medium. Chromaffin granules were prepared from bovine adrenal medullary tissue by differential contrifugation in 0.3 M sucrose (1). Ten mls of purified granules ('100 mg protein) were lysed by dilution with 10 volumes of deionized water and the membranes sedimented at 48,000 g for 30 min. The membranes were resuspended in a second 10 volumes of water and centrifuged again. The washed membranes (-20 mg protein) were resuspended in 10 mls of 0.5 M NaCl, 0.1 M NaHC03 (coupling buffer) and mixed with a slurry of CNBr-activated Sepharose 4B prepared from 10 gms of powder according to the manufacturer's recommendations (Pharmacia). Coupling of the membranes to the gel beads was permitted to proceed for 2 hours at room temperature on a shaking table followed by incubation at 4oC overnight. The gel was then washed three times with coupling buffer and incubated for 1 hr at room temperature with lM ethanolamine, pH 8.0. Chromatography. The affinity gel was washed by gravity sedimentation 3 times in 10 volumes of column buffer (240 mM sucrose, 30 mM KCl, 32 mM histidine-HCl (pH 6.0) and packed into a 1.6 x 10 cm water jacketed column. The column temperature was maintained at 370C during chromatography. The column was washed with 2 bed volumes of column buffer including 4 mM CaC12 and 2 mM EGTA, followed by two volumes of column buffer including only 2 mM EGTA. This washing cycle was repeated once and the column finally equilibrated with buffer containing 4 mM CaC12, 2 mM EGTA. A cytosol fraction was prepared from adrenal medullary tissue by recentrifuging at 100,000 g for 1 hr the initial 20,000 g supernatant obtained during the preparation of chromaffin granules (1). Solutions of KCl, histidine.CaC12 and EGTA were added to the cytosol, which was prepared in 0.3 M sucrose, to match the column buffer including 4 mM CaC12, 2 mM EGTA. Twenty mls of cytosol containing approximately 236 mg of protein were applied to the column followed by two column volumes of buffer including 2 mM free Ca2+ (4 mM CaC12, 2 mM EGTA). To elute bound proteins, the free Ca2+ concentration of the column buffer was reduced in two steps, to 50 uM, then 0.1 pM, by reducing the CaC12 concentration in the presence of 2 mM EGTA (2). Analytical Methods. Electrophoresis was carried out in a 10% polyacrylamide slab gel in SDS according to Laemmli (3). TWO and one half ml fractions of the column eluate were prepared for electrophoresis by buffer exchange on Sephadex G25 into 50 mM NH4HC03 followed by lyophilization and resuspension in electrophoresis starting buffer. Protein was assayed by the method of Bradford (4) with bovine serum albumin standard. Chromaffin granule aggregating activity of the column eluate was determined as in the standard assay for synexin activity (5), by measuring the turbidity (A 540) increase induced by 400 ul fractions of the eluate in a one ml chromaffin granule suspension at 370C in the presence of 1mM free Ca2+.

1396

Vol. 103, No. 4,198l

BIOCHEMICAL

AND BIOPHYSICAL

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Figure 1. Protein content and chromaffin granule aggregating activity of the affinity column eluate. The bulk of the cytosol passed through the column and was collected in fractions 19 through 24. Flow rate 1.3 mllmin, fraction size 3.5 ml. Protein content is represented as solid line (left axis), and aggregating activity as dashed line (right axis) the change in turbidity of a.chromaffin granule suspension representing induced by 400 ul of eluate. The changes in Ca*+ concentration of the eluate are marked by the arrows. Figure 2. Electrophoretic analysis of proteins in the affinity column eluate. Molecular weight standards are in lane "5" and are marked in kilodaltons. Lane "W" contains proteins washing from the column before reduction in [Ca*+] (fraction 37, fig 1). Lane "50" contains proteins eluting 39, fig 1). Lane "0.1" contains proteins eluting at 50 yM Ca*+ (fraction at 0.1 uM Ca*+ (fraction 51, fig 1).

RESULTS The protein the affinity duction eluate

column

of free

might

have

the membranes.

amount

of additional

maffin

granule

granule

and chromaffin

eluate

Ca 2+, from

was completely

which for

content

led

free

illustrated

of protein loss

Immediately protein

activity

aggregating

in figure

1:

2 mM to 50 PM, was introduced

to the

aggregating

aggregating

are

granule

in order

of proteins following

appears activity

in (fig.

was eluted

1397

to avoid that

the

1). when

which

Additional [Ca2+]

The first

of re-

before

the column

extensive

washing

have only

reduction

the eluate

activity

weak affinities

in

[Ca2+],

displays protein

was further

a small chrowith

reduced

to

Vol. 103, No. 4,198l

0.1 uM (fig.

BIOCHEMICAL

1).

The total

was 93 ug, or 0.04% 2 illustrates eluate. ing

washed

eluted 0.1

"WI' is

from

when

51,

proteins

In addition,

lane

appear

and at 0;l

in

the proteins

The interpolated

molecular

which

were

lane

and to

weights 36,000,

of 34,000,

and 33,000.

of proteins

primarily

This

u5Ou)

46,000,

44,003

be-

specifically

39, lane

uM are: 70,000,

proteins

was reduced.

60,000,

Figure

the column

and represents

to consider

amounts

procedure

column.

to 50 uM (fraction

uM Ca2+ small

and 32,000,

to the

[Ca*+]

at 50 uM Ca2+ are

3 illustrates

absorbtion

The most prominent

gel.

32,000

72% of the protein

obtained

in both

tained

The binding dependent

of molecular

eluted

weights

at 50 PM Ca2+,

also

by these

isolate cytosis,

of molecular

efficiency,

at 50 PM Ca*', amounts

these

weights

two peptides

and 57% of all

of the other

protein

proteins

ob-

3.

to the

granule

membranes

was specifically

obtained

were

2mM Mg2+ and 2mM Na2Al'P

with

and "0 . 1" of

"50"

those

in as much as the same results

experiment

in the absence procedures.

cytosol

clearly

to fig.

proteins

of lanes

when the

continuously

present

buffers.

In a control processed

staining

eluting

of these

was conducted

the column

equal

in the legend

on Ca2+,

experiment

are

The relative

steps.

are given

scans

bands

Assuming

and 34,000.

comprise

ing

which

by this

of the proteins

the

COMMUNICATIONS

in the eluate. Figure

in

was applied

before

"0.l").

eluted

at 0.1

34,000

just

was reduced

and 26,000,

60,000,

that

RESEARCH

obtained

37 of the eluate

against

[Ca*+]

of protein

analysis

fraction

the column

uM (fraction

the major

the

of the protein

a background

32,000,

amount

an electrophoretic Lane

provides

AND BIOPHYSICAC

of granule

When the

in 0.1

proteins

in which

the

membranes,

reverse

uM Ca2+ and eluting that

no proteins

might were

CNBr activated

become detached obtained.

1398

no proteins

experiment with

Sepharose

was

2 mM Cazi, from

were

performed

4B was

obtained of apply-

in an attempt

the granule

during

to exo-

Vol. 103, No. 4,1981

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

scans of lanes "50" (top) and "0.1" (bottom) of Figure 3. Densitometer the gel in fig 2. Interpolated molecular weights of the major proteins are marked in kilodaltons. Comparison of the areas under the peaks indicates: At 50 VU Ca2+, 93% of the stained protein is in the labeled peaks, 5% at 26,000, 39% at 32,000 33% at 34,000, 4% at 36,000, 5% at 46,000, 7% at 60,000; at 0.1 @I Ca2J, 77% of the stained protein is in the labeled peaks, 6% at 32,000, 28% at 33,000, 16% at 34,000, 8% at 44,000, 19% at 70,000; of the total protein eluted at both steps, 69% was eluted at 50 PM Ca2+.

DISCUSSION It

was anticipated

pendent

affinity

synexin

binds

46,000

molecular

synexin

should

that

synexin

chromatography to the

also

technique

granule

weight

membrane

protein

(The previously

reported

molecular

distinguishable

from

46,000

the interpolation

procedure).

and this

(6,7),

applied

eluted weight

Actin

by the Ca*+ de-

in this

study

since

manner

(5).

at 50 UM Ca*+ may be synexin

suggested

may be the 44,000

be isolated

in a Ca*+-dependent

eluting

be predominantly

the

would

also

of 47,000

(5)

here,

within

the limitations

binds

to the

granule

molecular

weight

as

Ca 2+ concentration.

at the higher of synexin

The

protein

is

inof

membrane

eluting

at 0.1

UM Ca*+. The other the power

proteins

of this

obtained

technique

are something

to bring

of a surprise,

to our attention

1399

previously

and illustrate unsuspected

Vol. 103, No. 4,198l

BIOCHEMICAL

interactions

between

occur

exocytosis.

during

some of these another

cytosolic

proteins

proteolytic

bind

as chromogranins These

functions

in the (8),

class

analogy

terms

membrane

be called

individual molecular

weights

synexin.

Now that

is

to make it

in exocytosis

nor

chromaffin

it

whether

is not

that

clear that

might

whether is,

-via

some of the proteins

granule

core

of the granule replaced

are

the proteins

are

that Rather

the term

obsolete

by learning

or other

activities

assigning

that

chromobindin

has been

secretory

their

introduced,

roles

these

A is in granule

letters,

to by including

the specific of the

chromomembrin

to the chromaffin

For example,

chromobindin

names when the

I suggest

bind than

known

as chromomembrins

by more precise For example,

may be referred

in kilodaltons.

collectively

membrane

of dopamine-B-hydroxylase.

chromobindins.

chromobindins

point

membrane

to the membrane,

become known.

form

these

at this

COMMUNICATIONS

of others.

names are being

the membrane-bound

RESEARCH

and the granule

indirectly

proteins,

and those

of the proteins

with

only

fragments

The proteins

(9).

proteins

Of course,

one of the eluted

merely

AND BIOPHYSICAL

the approximate

46 is the proteins

probably challenge play

vesicle.

ACKNOWLEDGEMENTS This study was initiated in the Intramural Research Program of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases. I thank my colleagues at the N.I.H., Harvey B. Pollard, Christopher J. Velia M. Fowler and Janet H. Scott for discussions and encouragePazoles, ment. The study was completed at the University of Virginia with funds from Biomedical Research Support Grant 5 SO7 RRO5431-20 and N.I.H. equipment loan 55-81. I thank David Sterner for technical assistance and Sharon Lipka for typing the manuscript.

1. 2. 3. 4. 5. 6. 7.

a. 9.

REFERENCES Pazoles, C.J. and Pollard, H.B. (1978) J. Biol. Chem. 253, 3962-3969. Caldwell, P.C. (1970) in Calcium and Cellular Function (Cuthbert, A.W., ed), MacMillan, London, 10-16. Laemmli, U.K. (1970) Nature 227, 680-685. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Creutz, C.E., Pazoles C.J. and Pollard H.B. (1978) J. Biol. Chem. 253, 2858-2866. Burridge, K. and Phillips, J.H. (1975) Nature 254, 526-529. Fowler, V.M. and Pollard, H.B. (1982) Nature, in press. Blaschko, H., Comline, R.S., Schneider, F.H., Silver, M., and Smith, A.D. (1967) Nature 215, 58-59. Winkler, H. (1971) Phil. Trans. R. Sot. Ser. B 261, 293-303.

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