Characterization of KEX2 -encoded endopeptidase from yeast Saccharomyces, cerevisiae

Vol. 159, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

February 28, 1989

CHARACTERIZATION

Kensaku

OF KEX2-ENCODED ENDOPEPTIDASE SACCGMYCES CEREVISIAB

MIZUNO, Tomoko Shoji TANAKA*,

Department

January

4,

FROM YEAST

NAKAMURA, Takehiro OHSHIMA*, and Hisayuki MATSUO

of Biochemistry, Kiyotake, Miyazaki

Miyasaki 889-16,

*Suntory Institute for Shimamoto. Mishima, Received

305311

Medical Japan

College,

Biomedical Research, Osaka 618, Japan

1989

SUMMARY: Yeast Saccharomyces cerevisiae KBXZ gene previously isolated was characterized as the gene encoding an endopeptidase required for proteolytic processing of precursors of e-factor and killer toxin. In this study, the cloned KEX2 gene was introduced into the kex2 mutant cells and the KEXZ gene product expressed in these cells was parEly purified from theirzrane fraction. The enzyme preparation exhibits a calcium-dependent endopeptidase activity with a substrate specificity toward the carboxyl side of Lys-Arg, Arg-Arg and Pro-Arg sequences. The enzyme activity was inhibited by serineprot.ease inhibitors, such as DFP and PMSF, indicating that the KEXZ endopeptidasn belongs to a serine-protease family. The optimal pH was determined to be around 5.5. the KEX2 endopeptidase was found to be a Thus, unique calcium-dependent serine-protease distinct from calpain and trypsin. 0 1989 Academrz Press, Inc.

Many bioactive as

t.heir

own

precursors,

proteolysis, known

about

processing identified and

killer

deficient specificity indicating

peptides

generally the

and secreted which

a.t sites

responsib1.e

are

precursors

kex2

mutants

toward that

the

processed

of paired

restored

the

carboxyl

KEX2

gene

product

forms

of

involved

the

of cloned

activity paired

may be

by

However,

processing

of

synthesized

cerevisiae,

processing side

mature are

Saccharomyces proteolytic

initially

residues.

that

Introduction

(3).

are to

basic

endopeptidases

(1,2). In yeast --in vivo as the gene required for toxin

proteins

basic

an endopeptidase

limited

little

iS

in

precursor

KEX2

gene

o-mating KEX2 with

was

factor gene

into

hydrolytic

residues involved

(3,4), in

Abbreviations: MCA, 4-methylcoumaryl-7-amide; AMC, 7-amino-4-methylcoumarin; Boc-,t-butyloxycarbonyl-; PMSF,phenylmethylsulfonyl fluoride; DFP,diisopropyl fluorophosphate; pAPMSF,p-amidinophenylmethylsulfonyl fluoride; TLCK,p-tosylB-ME, 2-mercaptoethanol; DTT, dithiothreitol; L-lysine chloromethyl ketone; ethylene glycol bis(8-aminoethyl ether)-tetraacetic acid; STI, soybean EGTA, trypsin inhibitor; pCMB, p-chloromercuribenzoate. UCIO6-291X/89 $1.50 305

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 159, No. 1, 1989 precursor

processing

unique

and

dependency, with

Enzymes partially the

was

characterized

to

be

of

the

resembling sequence of

the

to the members the molecular we

protein, introduced

with

that optimal

calpains

KEX2-encoded

studied was the

its

the kex2 partially

KEX2

and

gene

contains

properties. and

purified

and is

to

the

residues

(3,4).

endopeptidase

tissues

(8).

showed

that

were

studies

thiol-dependent

(3-7), neutral

However, the

recent

amino

acid

extensively

homologous

family (9,lO). the action of

To clarify KEXZ-encoded

The KEX2 gene

cloned product

characterized.

a unique

exhibit calcium-

In these

a region

enzymatic cells

found

basic KEXZ

(7).

a calcium

serine-protease proteolytic

KEX2 endopeptidase

pH at around

the

and ours

the

mutant

paired of

from mammalian

protein

of subtilisin-like mechanisms of

into

cells

the

analysis

those

(S,6)

was

membrane-association, toward

to

group

nucleotide

reveals

specificity similar

by Molf's

protease,

these

including

substrate

properties

RESEARCH COMMUNICATIONS

KBXZ endopeptidase

purified

enzyme

sequence

The

--in vivo. properties,

enzymatic

AND BlOPHYSlCAL

Ca

2+

-dependent

The

KEX2

gene

was

expressed

in

present

study

serine-protease

5.5. MATERIALS

AND METHODS

strains and plasmid: Saccharomyces cerevisiae ~16-57C (MATa leu2 w ura3 kex2-8) was used for the kex2 mutant (10) and X2180-1B usedforxd type. The plasmid pYE-KEX2(5.0)b, carrying the 5.0 Kb EcoRI-Sal1 fragment of KEX2 gene (named KBX2(-), was used for transformation (10). Medium andgrowth conditions: Yeast cells were grown at 30°C in SDCA medium (0.7% Bacto-yeast nitrogen base-2% dextrose-2% casamino acids), supplemented as necessary with tryptophan (20 ug/ml). Preparation of enzyme: Yeast cells (wet weight ca. lg) were harvested by centrifugation, washed once with 0.9% NaCl and resuspended in 5 ml of 0.1 M sodium phosphate buffer (pH 7.4). They were lysed by incubation with 2.5 mg of Zymolyase-100,000 (Seikagaku Kcgyo) at 37'C for 2hr, and homogenized with a Polytron homcgeneizer. After centrifugation at 80,OOOg for 20 min, the pellets were washed four times by resuspension in 5 ml of 10 mt+l Tris-HCl buffer (pii with centrifugati0.n each time at 80,OOOg for 20 min. The pellets thus 7.0), obtained were extracted twice wi.th 5 ml of 10 mM Tri.s-HCl (pH 7.0) containing 0.2 M NaCl and 1% Lubrol (PX type, Nakarai Chemicals). After centrifugation at 80,OOOg for 20 min, the supernat.ants were used for measurement of solubilized membrane-associated enzyme activity and protein concentration (11) (Table 1). For further purification, the membrane extracts were desalted with PD-10 column (Pharmacia), equilibrated with 10 mM Tris-HCl (pH 8.0)-0.2% Lubrol, and applied to a column of DEAE-Sepharose CL-6B (1.4 x 44 cm). The column was eluted with a linear gradient from 0 to 0.5 M NaCl in 10 mM Tris-HCl buffer (pH 8.0)-0.2% Lubrol (Fig.1). The active fractions were pooled and used for characterizing their enzymatic properties. Enzyme assay: Protease activity was measured as follows (7,lO): Twenty nanomoles of Boc-Gln-Arg-Arg-MCA were incubated at 37OC with the enzyme in 250 ul of 0.2 M Tris-HCl buffer (pH 7.0) containing 0.1% Lubrol and 1 mM EGTA in the presence or absence of 2 mM CaC12. In the experiments to characterize the anzymic properties, 0.2 M Tris-HCl buffer was replaced by 0.2 M acetate buffer (pH 5.5). The amounts of AMC released from the substrate were measured by a fluorescence spectrophotometer with excitation at 380 nm and emission at 460 endopeptidase activity was evaluated after the addition nm. Calcium-dependent of an excess amount of CaCl . One unit of enzyme activity is tentatively defined as the amount of enz&e that can release 10 nanomoles of AMC from the substrate under the above assay conditions in 1 hr. 306

Vol. 159, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION The capable

5.0

Kb

of complementing

(10).

Introduction

killer

activity

a specific membrane

cells),

Lubrol

and

comparison, wild

remarkable the

assayed

for

cells

Sephrose

CL-6B

the

the

membrane

1C).

(Fig.

1).

endopeptidase

KEX2 gene

introduced

appears

to

expressed

in

Fig.

were

lB,

obtained

from

enzymatic

in

the

Table

cells

(Fiq.

from

activity

type type

kex2

the cells.

The

active in

eluted similar

cells

expressed to

portions, this

36-40

those

of

1B and calcium-

product

of

based

the

on the

in

KEX2

cells

of

the

enzyme

in

Fr.

36-40

lC,

enzyme

exhibited from

KEX2

its cells

below.

1..

Calcium-dependent

endopeptidase activity yeast cells Total

Strain

(1)EGTA

activity (2)BGTA+Ca

(units)

(2)- (1)

Kl6-57C

5.2

5.4

0.2

1116-57C [pYB-KF.X2(S.O)bl

3.2

19.8

16.6

X2180-U

8.5

16.9

8.4

in

membrane extracts Total protein (mgl 23.7 9.73 14.4

of

Specific activity (units/w) 0.01 1.71 0.58

The membrane extracts were prepared as described in "MATERIALS AND METHODS". was measured by using Boc-Gln-Arg-Arg-MCA as a Bndopeptidase activity Total protein was measured by the method of Lowry et al. (11). substrate. 307

in

The enzyme preparation

in Fig. the

in

membrane

the

those eluted

study.

DF,AF,-

position

in the

Furthermore,

similar

of

(Fig.

that the

a

activity

cells

cells.

in Fr. to

is

in

extracts

elution

product

properties

and characterized very

KEX2

gene

the

indicate

a

encodes

membrane

type

1,

was observed

was detected

facts

deficient

KEX2

in

mutants

Table

endopeptidase

activity in

in

on a column

of wild

These

found

cells,

the

For

kex2

the KEX2 gene

observed

extracts

1A).

the

that

detergent

activity.

extracts

chromatographed

endopeptidase

pooled wild

1% non-ionic

analyses,

was

membrane

physicochemical

wild

were

both

of cleaving

as reported (10). The carrying pYE-KF,XZ(S.O)b

calcium-dependent

KEX2

into

properties

characterized

cells

behavior, have

regained

capable

endopeptidase

further

A major of

mutants

dependent

chromatographic

reported

kex2 mutants

containing

indicating

For

respective

kex2

as previously

activity

of the membrane

of KEX2 cells,

to that of

a buffer

KEX2 (5.0))

also measured. As clealy seen 2+ in a Ca -dependent endopeptidase activity

No calcium-dependent

extracts

the

(named

were

extracts

corresponding

into

endopeptidase

with

endopeptidase.

from

gene

was isolated

K~X2(5.0)

activities

extracts

obtained

KEX2

calcium-dependent

relative

Ca2+ -dependent

the

substrate (Boc-Gin-Arg-Arg-MCA), prepared from the transformants

increase

membrane

cloned

extracted

type

of

mutation

Ca2+ -dependent -

were the

fragment

the kex2

of the and

fluorogenic fractions,

(KEXZ

and

EcoRI-Sal1

Vol. 159, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(A)

50 - 150

1.5 00

- 100

1.0 IO

- 50

0.5 I

0

I

I

J

I

I

-0

L

1.5

1.c

I. 2

Es

0.:

0 (Cl

I---__-___

1.0

I

l.! i-

I

0.5

_----J ___---

1.1I-

__

--

__-_-_--_e-

--

___---_------

0

I 0

j

Ai

O.! S-

I

I

I

I

I

10

20

30

40

50

Fraction

number

1

I 60

I

70

(4 ml/fr.)

DEAE-SepharOSe CL-68 chromatography of the solubilized membraneFig. 1. fractions obtained from (A) Kl6-57C (kexl mutant), (B) K16-57C[pYE-KEX2(5.0)b] (transformant with KEXZ(5.0) gene), and (C) X2180-1B (wild type). Aliquots (150 (11) from each fraction were assayed for endopeptidase activity at 37'C for 6 hr in the presence or absence of an excess amount of CaC12, as described in "MATERIALS AED METHODS".

To

characterize

various

fluorogenic

amounts

of

AMC

spectrophotometer containing

Arg-Arg basic

rate. the

The carboxyl.

enzyme side

substrate

specificity

MCA-derivatives released

from

(Table

of

paired

the

and

2).

also of

As

Lys-Arg

residues,

residue.

two

were were

substrates On

the

308

hydrolyzed some containing

other

the

measured

reported

there

KEX2-encoded

with

were

previously

sequences

the

incubated

substrate

although cleaves

Arg

were the

of

hand,

enzyme, by

(7), on

a

and

the

fluorescence

MCA-substrates the

differences Pro-Arg typical

protease,

carboxyl in

side reaction

sequence substrates

on for

Vol. 159, No. 1, 1989

BIOCHEMICAL

Table

2.

Substrate

Substrate ioc-Gln-Arg-Arg-MCA Boc-Leu-Arg-Arg-MCA Boc-Gly-Arg-Arg-MCA Boc-Leu-Lys-Arg-MCA Boc-Gly-Lys-Arg-MCA Boc-Val-Pro-Arg-MCA Boc-Ala-Pro-Arg-MCA

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

specificity

of

% Activity 100.0 116.2

KEXZ endopeptidase

Substrate BOC-Glu-Lys-Lys-MCA Pro-Phe-Arg-MCA Z-Phe-Arg-MCA Bz-Arg-MCA Arg-MCA Leu-MCA

11.9 92.8 59.2

38.1

% Activity E 0:9 0.7 0.9 1.0

17.0

Protease activity for Boc-Gln-Arg-Arg-MCA was taken as 100%. Each substrate (20 nmole) was incubated with the enzyme at 37°C for 4.5 hr as described in "MATERIALS AND METHODS". AMC released was measured by fluorescence spectrophotometer.

trypsin

and kallikreins

aminopeptidase Lys-Lys

substrates

seqence

findjngs

indicate

specifi.city

that

toward Thus,

at these

signals,

the

the

three

optimal optimal vesicl.es vesicles

enzyme

paired

basic

enzyme

is

activity

and

a substrate

affected

by the

exhibits

the

(Lys-Arg,

containing

(12),

killer

These

substrate and

in proteolytic

of a-factor

two

enzyme.

unique

Arg-Arg)

to be involved

in precursors

pH of

was reported buffer

5.5

(141,

systems

Pro-Arg

processing toxin

is

consistent

Table

In previous

to be around

(13)

with

that

the

the

internal is

3 summarizes

the

.r4> 'Z 50 4" *

.-3 5504" *

O7

8

9

10

-7

(3-y).

and

pH actually

effects

the

optimal by using

glycine-NaOH),

the

5.5

The

found

(Fig.ZA). in

secretory

operative

of various

in

reagents.

(B)

I -6

0 -5

I -4

lag

PH

examined

However,

to be around

enzyme

loo-

6

7.0

were

studies,

Tris-HCl,

determined

100

5

on enzyme activity

(AcONa-AcOH,

was

suggesting

vivo. -

reagents

as a substrate.

enzyme activity

for

0 4

not

residues

likely

of pH and various

different

--in

Leu-MCA) were

KEXZ endopoptidase

Boc-Gin-Arg-Arg-MCA

pH

and

and Z-Phe-Arg-MCA),

proteins.

The effects pH for

the

found

secretory

bp using

(Arg-RCA

Pro-Phe-Arg-MCA

(HOC-Glu-LYS-LYS-MCA)

secpence. other

(Bz-Arg-MCA,

[Cal

-3

-2

Three buffer systems (sodium2. (A) Effect of pH on enzyme activity. and glycine-NaOH (-&-)) were used at 0.2 acetate ( +), Tris-iiC1 (+), EGTA-CaCl M. (B) Effect of calcium ion concentration on enzyme activity. I Free calciun& concentration was calculated by using buffers were used. dissociation constant of 4.2 x 10 M (20).

Fig.

309

the AS

BIOCHEMICAL

Vol. 159, No. 1, 1989

Table

3.

Effects

of

various Activity

Effector (A)

control(+0.5mM DFP

CaC12) mM mM mM mM mM mM mM mM mM 1:

1 3.3

10 1

PMSF

3.3

10 1

pAPMSF

3.3

10 TLCK

1 10 1 10

ICH2COONa

mM mM mM mM mM

1 10

ICH,CONHz pCMB DTT B-ME EOTA EGTA

:

1

z

1

mM mM mM

1 10

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reagents

on KEXP endopeptidase Effector

1 mM

100 92 93 5 98 3 1 82 0

ZnC12 HgClz ST1

10: 37 66 0 64 15 4

bestatin

mM 0.1 mg/ml 1 mg/ml 0.1 mg/ml 1 mg/ml 1mM 10 mM 1 mM 10 mM 1 mM 10 mM 1mM 10 mM

leupeptin pepstatin

A

E-64

control + 1mM + 2mM + 2mM + 1mM + 1mM

10; 404

6 1 94 75 103 96 24 3 113

1

Trasyrol

(B)

activity Activity

ii: 105 90 79

(none) EGTA CaCll MgC12 EGTA + 2mM CaClz EGTA + 2mM MgC12

100 9 316 72 321 9

5

Protease activity was measured as described in "MATERIALS AND METHODS". The enzyme was preincubated with the reagent at 30°C for 30 min in the presence of 0.5 mM CaC12 (A) or in the absence of CaClz (B). In the reactivation experiments in (B), the enzyme was inactivated with 1 mM EGTA at 30°C for 30 min, and then incubated with CaC12 or MgC12 as above.

previously

reported

protease

(3-7),

inhibitors,

concentration

(10 mu) of

completely

inhibited

typical

Thus,

protease

Recent

KEX2 endopeptidase

suggest similar

s cystein

in the

family

are that to

well

the

active

site

The enzyme EGTA was restored of

l.ocated

may

reagents,

into

immediately

due

showed

around

the

active

together,

(lo), the

blockage

adjacent

that

to subtilisin-like

taken

to

enzyme

a serine-

of KEX? gene

previously be

En2+),

on

as a serine-protease

As discussed

by thiol-reagents (Cys-217)

findings,

was

(Bg2+,

no effect

homologous sequence

function

activity

ions

classified

analysis

acid These

may

of subtilisin.

that

amino

had

be

extensively

(9,lO).

KEX2 endopeptidase

residue

addition

and

enzyme

metal

serine-

by

At a higher

by thiol-directed

may

sequence

inhibited at 1mM.

the

E-64,

endopeptidase a region

conserved

endopeptidase

however,

pCME, and heavy

nucleotide

contains

serine-protease residues

reagents,

thiol-protease,

KEX2

not

was

and pAPMSF,

The enzyme was inhibited

for the

family.

activity

PMSF,

iodoacetaaide,

inhibitor

activity.

enzyme

these

(9).

such as iodoacetate, but

the

such as DFP,

site

strongly a manner

in

inactivation of the

to a histidine

of free

residue

(10). was also by

the

inhibited addition

by EDTA and ECTA. The inhibitory of an excess

amount

of CaC12,

concentration

(Table 3(B)). MgC12 on enzyme activity.

The enzyme was half-maximally

approximately

10 nM Ca'+.

-KEX2

Thus,

Fig.fB

310

shows

endopeptidase

tne

but

effect

appears

effect not of

by the calcium

activated to

of

at

be a unique

Vol. 159, No. 1, 1989

calcium-dependent

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

but

serine-protease,

not

related

to calpains

and pancreatic

trypsin. Paired

basic

of various from from

to

rat

described

in

are

this

to

and

that

mature

liver into

mature

endopeptidases

may be involved

with

were

Although

species which

characterized

it

is

not

family

peptides properties

showed to

(19). similar

processing

clear

or

not,

substrate

with

report

was able

in precursor

eukaryotic

(membrane-association,

recent cells

processing

endopeptidases, products,

pH optimum)

Furthermore, in mammalian

of

a serine-protease

properties and acidic

of

range

(17,lE).

classified

a set

of proteolytic

calcium-dependent

some overlapping study.

sites

i.n a wide

to their

(15,16)

expressed

opiomelanocortin endopeptidase

Recently,

enzymes

as the

precursors

calcium-dependency

product

possibility

known

and proalbumin

seem to have

specificity, gene

mammals.

insulinoma these

are

peptide

proinsulin

whether they

bioactive

yeast

convert

residues

that

These to

yeast process

correctly

facts those

in mammalian

KEX2 pro-

suggest of

the

a KFX2

tissues.

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