Targeting aequorin to the endoplasmic reticulum of living cells

Vol. 189, No. 2, 1992 December 15, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1008-1016

TARGETING AEQUORIN TO THE ENDOPLASMIC RETICULUM

Jonathan

M. Kendall,

Department of

Robert

of Medical Medicine,

L.

Dormer

Biochemistry, Heath Park,

OF LIVING

and Anthony

K.

CELLS

Campbell

University of Wales Cardiff CF4 4XN, UK

College

Received November 2, 1992

SUMMARY: The photoprotein aequorin has been engineered with an ER targeting sequence at the N-terminus, with and without KDEL at the C-terminus, so that it locates in the ER-secretory pathway. For the first time the free Ca2+ has been quantified inside the ER and shown to be 5-20 times that in the cytosol. In COS cells free Ca2+ in the ER ranged from l-5mM at 37oC, decreasing 2-5-fold within lmin of exposure to the Ca2+ 0 1992Academic ionophore ionomycin in the absence of external Ca2+. Press,Inc.

INTRODUCTION: internal

Ca2+ store

calcium

in

may be released

external and are

The endoplasmic

stimuli

reticulum(ER)

eukaryotic into

role

the

or pathogens.

cytosol

ADP ribose, of

these

the

stores

other in

Signal

peptides

direct

by Ca2+ it to

living tells(7). Those destined membrane or secretory vesicles ER, and not terminus(8,9).

sequence. also secreted, Our

Ca2+-activated photoprotein the ER by engineering signal aequorin, one in the ER(lO), proteins 0006-291X/92

to

is live

particular

which

response

to

Ca2+ oscillations there or the

localised

Ca2+ that

sites

plasma to the ER

directed

was to

free

within

lysosomes,

to be retained a KDEL sequence

strategy

within at the

locate

the C-

the

aequorin as a Ca2+ indicator inside sequences onto the N-terminus of a major Ca2+-binding protein

$4.00

Inc. reserved.

from

now essential cells.

from calreticulin, and one fromp-lactamase(ll), The efficacy isolated microsomes.

0 1992 by Academic Press, of reproduction in any form

major

To elucidate

for the ER, are initially

Proteins contain

experimental

(2,3). waves,

ER in

proteins

the

the

has been proposed that one releasable by IP3

oscillations,

by an N-terminal

in

To explain

transients, and disease(4,5,6), Ca2+ is measured inside the

Copyright All rights

cells(l,2),

waves seen in many cells, it at least two distinct stores,

cyclic

is

1008

which directs of these peptides

Vol.

189, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

in targeting aequorin to the ER was tested first using isolated microsomes. Normal aequorin and aequorin with the calreticulin or f3-lactamase signals with or without KDEL were then expressed in COS cells. Light emission from aequorin reactivated within the live cells then enabled the free Ca2+ inside the ER and cytosolic compartments to be measured independently. MATERIALS

AND METHODS

to aminoDNB: Aequorin DNA, equivalent acids 1-189 of the full sequence(l2,13), was isolated by PCR of gDNA, and extracted from freeze dried Aequorea Victoria collected at Friday Harbor, Washington State(kind gift from Dr C.C. Ashley, University of Oxford). The DNA was engineered by the polymerase chain reaction (PCR) using the following oligonucleotide primers:AEQ5: TAATACGACTCACTATAGGGAGAATGGTCAAGC TTACATCAGACTTCGAC (5'end sense including T7 promoter); AEQ7: CCATGTCGACTTAGGGGACAGCTC (3'end antisense including SalI restriction site);AEQ28: GTGCCGCTGCTGCTCGGCCTGCTCGGCCTGGCCGCCG CCGCCGTCAAGCTTACATCA (5'end sense); AEQ27: CACCTAATACGACTCACTA TAGGGAGAATGCTGCTCCCTGTGCCGCTGCTGCTC (5'end sense including GTCGCCCTTATTCCCTTTTTTGCGGCATTTT clamp and T7 promoter);AEQ30: GCCTTCCTGTTTTTGCTGTCAAGCTTACATCA(5'end sense): AEQ29: CCTAATA CGACTCACTATAGGGAGAATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCC (5'end sense including clamp and T7 promoter); AEQ9: GTCGACTTACAGCTCA TCCTTGGGGACAGCTCCACCGTA (3'end antisense including clamp and Sal1 restriction site). The T7 RNA polymerase promoter was added to the 5' end of apoaequorin gDNA and a Sal1 site to the 3' end (AEQN) using primer and AEQ7 oligonucleotide primers AEQ5 as the 5' sense In order to generate DNAs extended as the 3' antisense primer. at their 5' ends with sequences encoding ER targeting or at the 3' end by the seqence encoding KDEL, two sequences, stage PCRs were employed (14,15,16). Apoaequorin extended by: (a) the p-lactamase signal (AEQB): MSIQHFRVALIPFFAAFCLPVFA(ll), was obtained by using AEQ28 and AEQ7 in the first stage and AEQ27 and AEQ7 in the second stage PCR; (b) the calreticulin signal (AEQER): MLLPVPLLLGLLGLAAA (lo), by using AEQ30 and AEQ7 in the first stage and AEQ29 and AEQ7 in the second stage. Apoaequorin DNA extended at the 5' end with the sequence encoding the calreticulin ER targeting sequence and at the 3' end by the sequence encoding KDEL and a Sal1 site (AEQERK), was obtained by PCR of AEQER with AEQ29 and AEQ9. Successful PCR was confirmed by the sizes of the fragments on agarose gel electrophoresis. Recombinant normal and modified apoaequorins were generated by first transcribing the PCR DNA products with the T7 RNA polymerase. The capped mRNA was translated in a nucleasetreated rabbit reticulocyte lysate system containing 35Smethionine in the presence or absence of canine pancreatic microsomal membranes. Reactions were either diluted in reducing SDS sample buffer and equal volumes electrophoresed, or reconstituted by addition of 1uM coelenterazine to measure chemiluminescent activity (16). . . COS-7 cells. t aem in - PCR products AEQN, AEQB, AEQER and AEQERK were cut with SalI, ligated into pSV7d cut with SmaI and SalI (17) and used used to 1009

Vol.

189, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

transform K12-MC1061 competent E.coli. Positive colonies (designated codes pSVAEQN, pSVAEQB, pSVAEQER and pSVAEQERK) were selected by PCR and the activity from these clones was investigated using in vitro transcription-translation to verify that PCR followed by sub cloning had selected active proteins. The COS-7 cell line was grown in DMEM medium (containing 10% foetal calf serum, penicillin and streptomycin) on plastic petri dishes. The plasmids pSVAEQN, pSVAEQB, pSVAEQER and pSVAEQERK were used to transfect the cells using the calcium phosphate method. After 48h, cells were detached from the petri dishes by treatment with trypsin, washed and resuspended in DMEM medium containing 2.5uM coelenterazine. Cells were incubated overnight in this medium at 4oC in the dark. Prior to each experiment, aliquots of the cells were centrifuged, washed and resuspended in a modified Krebs-Ringer Hepes buffer (KRB) (containing in mmol/litre: NaC1,120; KC1,4.8; KH2P04,1.2; MgS04,1.2; EGTA,l; HEPES,25, pH7.4 at 37oC), and transfered in a tube to the luminometer, maintained at 370C (18). Chemiluminescent counts were recorded either in the presence or absence of 1mM CaC12 and, where indicated the calcium ionophore (2uM ionomycin) was added. The remaining active photoprotein was estimated by lysis of the cells with 1% Nonidet in the presence of 25mM Ca2+. The rate constant of the aequorin decay was then estimated at various times from the equation (counts per second)/(remaining active photoprotein) as previously described(l8).

RESULTS

red mRNA in

rabbit

pancreatic the

microsomes but

not

bv mlcrosomes:

lysate

in

demonstrated or

native

was an increase

AEQER(calreticulin

DISCUSSION

aeqvorln

reticulocyte

calreticulin(AEQER)

terminus, there

AND

Translation

presence

processing

S-lactamase(AEQB)

the

signal)

molecular

, , , WACTAMASE

at

with the

N-

Surprisingly

weight

,

aequorin

signals of

and AEQB(B-lactamase

I , , CALRETICULIN

of

of

of

aequorin(AEQN)(Fig.l). in

SIGNAL

the

;

,

both signal)

from

McJl~~mass

-106 -80

z

MEMBRANES

pancreatic

1.

Processing microsomes.

L

-

+

of

-

+

recombinant

-

-

49.5

-

32.5

-

27.5

-

18.5

+

aequorins

by

isolated

DNA transcripts were synthesised using the T7 RNA polymerase and translated in a rabbit reticulocyte cell free system +/canine pancreas microsomal membranes. Translation products were analysed by electrophoresis on 15% SDS polyacrylamide gels. -+ represent the main aequorin bands. 1010

ca

Vol.

189, No. 2, 1992

BIOCHEMICAL

22.6kDa

to

24.8kDa,

21.8kDa

measured

Aequorin

contains

aequorins

RESEARCH COMMUNICATIONS

as opposed to theexpected for native aequorin without no consensus

thus the molecular basis modification is unknown reacted

photoproteins

AND BIOPHYSICAL

glycosylation

of this post-translational at present. However, the

with

with

N-linked

drop back any signal.

coelenterazine

kinetic

to

properties

form

to

the

sites, modified

apo-

Ca2+-activatable

similar

to

native

aequorin. .

n the

in the ER was established subcellular fractionation in

the

the

absence

of

constructs

apoaequorin estimated

by secretion and the effect

external

Ca2+.

pSVAEQER or per

culture

after

ER: The ability

COS-7

in

well

cells

containing

reactivation

with

of

the

with cells

constructs

into

four

Cal07

100,OOOg transfected supernatant(ie recovered

with in

showed

of

particulate

fractions

efficiency of targeting during fractionation. the

calreticulin

and

targeting aequorin that KDEL held the

because Together,

was

Fractionation

(nuclear=lOOOg,

the

apoaequorin in the up to

from

with pSVAEQER or pSVAEQB. This confirmed had occured. However it was not possible

48h,

and cytosolic=

pSVAEQN was recoverable it was cytosolic), whereas

the

in

or no from cells

fractions

85-96%

of KDEL, ionomycin

This

pSVAEQN.

microsomal=100,000g

supernatant)

cells

Little fluid

pSVAEQERK or

aequorin

transfected with approx. lOO-300ng

coelenterazine.

subcellular

mitochondrial=12,000g,

locate

the absence of ionophore

pSVAEQB secreted

some ten times that found in the cells. apoaequorin was detected in the culture transfected

to

in

cells

100,OOOg 70% was

cells

transfected

targeting to to determine

the the

ER

of the problem of ER damage these data confirmed that both

p-lactamase

signals

to the ER-secretory aequorin within the

were

capable

of

system in COS cells and ER, preventing it being

secreted. Active

aequorin

could

expressed apoprotein, up to 24h outside the terminal

proline

of

(21), and it was thus KDEL to the C-terminus However generate

the present sufficient

be formed

within

by addition cells. It

of coelenterazine(l8-20)for has been reported that

aequorin

is

essential

anticipated of aequorin data light

demonstrate emission 1011

live

cells

for

light

from

the the

C-

emission

that adding peptides such would destroy activity. that aequorin-KDEL for use in live cells.

can

as

Vol.

4

189, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

1A

RESEARCH COMMUNICATIONS

1B

cY-rosoL (pSVAEQN)

3 ? z x

2

B

\

1

0

ZO-

t N 2A

::I,

SECRETORY PATHWAY (PSVAEQER)

28

15?J 2 x loB

5-

OA

t KRB

t N

3A

30

I

1 minute

I

EiQure 2, Effect of the calcium ionophore, calcium in the cytosol and ER of COS7 aequorin luminescence. COS cells expressing 1) aequorin (AEQN), 2) and 3) ER-aequorin + KDEL (AEQERK) were Either A) Ringer buffer (KRB) without Ca2+. 1012

ENDOPLASMIC RETICULUM (pSVAEQERK)

ionomycin, on cells measured ER-aequorin incubated KRB or B)

in the

free by

(AEQER) Krebs Ca2+-

Vol.

189,

a

No.

2,

1992

BIOCHEMICAL

AND

BIOPHYSICAL

b

1mM EGTA

RESEARCH

IBM

COMMUNICATIONS

~a*+ ER-aequorin+KDEL ,/

EA;aequorin+KDEL

r -

.Ol ,/

z : zE

z +,

ER-aequorin

4

ER-aaquorin .

,001

Ei QI ;;; LT .OOOl

.OOOOI J,

I 20

0

30

40

.00001

50

J, 0

10

I

I

I

I

20

30

40

50

Time(s)

Time(s)

WF-3 . Aequorin chemiluminescence and rate constants in COS cells. COS cells expressing aequorin (U,m -AEQN), ER-aequorin (A,A-AEQER) and ER-aequorin + KDEL (0,. -AEQERK) were incubated in Krebs Ringer buffer (KRB) a) without Ca2+(lmM EGTA) or b) with 1mM Cap+ to measure the rate constant of aequorin decay as described in Methods, so that the absolute free Ca2+ concentrations in the ER could be calculated. Results represent the mean+/SEM of three determinations.

The

resting

200

times

in

the

glow that

of

cytosolic

in

some

or

extracellular immediate constant decrease

lo-20

to

of

correct

emanating

absence

of

Ca2+,

from

times

that

Ca2+,

addition decrease

fold

from cells in ER free

of

expressing Ca2+ from

the

3).

with

However,

0.2-3%

this

of

rate

constant

the

cells,

used

In

the

of

incubated

to

calculate

the cytosol, the ER or ER-secretory

cytosol.

the

consumption,

The

was then

even

ER-aequorin+KDEL

for

3.a,b).

(AEQN)

and the

compared

Ca2+(22) in either Ca2+ inside the

system.

5-20

order

decay

free

37oC,

estimated(Fig

chemiluminescent

the absolute ER-secretory was

In

were

at

80-90%

the

AEQERK was 100-

aequorin

EGTA(Figs.2

and

lmin

aequorin.

presence

native

extracellular

within

AEQER or

expressing

expressing

ER-aequorin

constants

the

cells

cells of

the

was consumed

the

of

presence

20-50%

rate

from

live

ER or

absence

the

system

of

of 2uM ionomycin resulted in an in the light emission and rate AEQER or ca 5w to

AEQERK, equivalent lpM(Fig.2;2A,2B,3A,3B).

to

ionophore ionomycin (I) was added (T) to measure the change in free Ca2+ in the ER compared with that in the cytosol. Remaining active photoprotein was estimated by lysis with 1% Nonidet in the presence of 25mM Ca2+ (N). Results are representative traces of chemiluminescence counts per second(cps) from several experiments. 1013

a

Vol.

189, No. 2, 1992

This

decrease

release addition

BIOCHEMICAL

in

light

intensity

of aequorin into of ionomycin to

ER signal

(AEQN)

reaching

in

within

could

a small,

5-lOs,

release

chemotactic in

the

peptide

absence

The lack

of

exposed

to

ER had

been

the

a small,

marked

+/-KBEL cells

of

external

higly

stored

for

A further

AEQERK) at several

higher

possible

for

from

ER when

the

the

than first

this the

minutes

In

longer

at

periods,

consumption. of aequorin

it

order will

affinity this

where

resting

Ca2+ neutrophils,

the

AEQER or targeting

cells

was

aequorin

of the fact that contained large

suggested

that

a temperature

ER had occured.

that

the

that

of to

free the

Ca2+ in cytosol.

measure

substantial

the It

directly

ER is

will

now be

Ca2+ release

Ca2+ influx

many physiological

to

AEQERK, to the

finding

expressing

from

outside

conditions(l,5).

for normal aequorin in the ER will be consumed within a few

follow

be necessary be achieved analogues

free to

Ca2+ inside reduce

responsible

this

the

for

ER for

aequorin

by using natural of coelenterazine

of aequorin for Ca2+(23,24). problem by generating a series

amino-acids

to

interesting

in spite 4oC still

at

means that photoprotein

This could and synthetic

reduce the have solved mutants,

370C.

is

under

As we have shown more than 90% of

37X, This

time

there

as occurs

in

hours

show clearly

reSultS

cell,

back

Ca2+(5).

consumption

(AEQER or

an

localised Ca2+ cloud with the phe or ionomycin, only detectable

efficient.

aequorin

by

Ca2+ transient

any Ca2+ spike in cells expressing ionomycin and EGTA, confirmed that

considerably

the

rapid

This cytosolic observed in human

highly fmet leu

amounts of active aequorin. dependent Ca2+ uptake into Our

be explained

and decaying

levels within 20-3Os(Fig.2;1A,lB). transient was similar to that which

not

RESEARCH COMMUNICATIONS

the external fluid. In contrast cells expressing aequorin without

resulted

maximum

AND BIOPHYSICAL

isoforms which can

However, we of aequorin

Ca2+ binding

have

been mutated to produce aequorin with a 20-fold reduced affinity compared to native aequorin(16). This mutant will be suitable for measurement of free Ca2+ in ER, endosomes, the Coelenterazine analogues are also mitochondria and externally. ratiometrically available enabling free Ca 2+ to be quantified Furthermore using a dual wavelength chemiluminometer(23,24). we have also shown that spectral changes in aequorin can be induced by mutating just a few amino-acids, thereby increasing the spectral range of the ratiometric indicators. 1014

Vol.

Measurements

of

plants(25,26), to of

BIOCHEMICAL

189, No. 2, 1992

free Ca2+ in together with shows

mitochondria(20), this technology.

for

The latter

visualising

targeting locations indicators fluorescent

ACKNOWB: discussions, preparations,

the

power

and

general to

single

a highly cells.

locate

molecular

cell

aequorin

biology,

the IP3 secretory

sensitive

method

The mutation

Ca2+ and protein kinase indicators(l5) live cells, opens up a new era for

in

aequorin

applicability

of the ER, including Golgi network, and

provide in

RESEARCH COMMUNICATIONS

in transgenic targeting of

now is

pools the

could

secretion

of in

E.coli and the recent

The challenge

specifically in different and Ca2+ releasable stores, vesicles.

AND BIOPHYSICAL

working

and

to specific bioluminescent in

symbiosis

with

indicators(27).

We thank Graciela Mr Andrew Trimby for and the MRC for financial

Sala-Newby help with support.

for the

helpful plasmid

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