Vol.
154,
No.
July
29,
1988
THE
2, 1988
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
tiQ.1OR
F’ROTEIN
OF PfiNCREQTIC
I)NCHORED
UIfi
Centre de Faculty
June
22,
ZYIIOGEH
COWLENT
Denis
Received
AND
BONDS
LeBel
recherche of Science, Sherbrooke,
and
GRFINULE
EIEliBRClNES
(GP-2)
818-823
IS
TO PHOSPHpTIDYLINOSITOL’
flarlyne
Beattie
les necanismes de secretion University of Sherbrooke DC, Canada, .JlK 2Rl
sclr
1988
SUMHRRY. GP-2, the major integral protein characteristic of the pancreatic zyaogen granule membrane can be released from the membrane by the action of a phosphatidylinositol specific phospholipase C (PI-PLCl , In a hydrophobic/ hydrophilic phase separation system using the non-ionic detergent Triton X-114, the aembrane-bound form of the protein went from the detergent phase into the hydrophilic phase upon action of the phospholipase. PI-PLC solubilization of GP-2 unmasked an antigenic determinant similar to the cross-reacting determinant of the trypanosone variant surface glycoproteins, This determinant being a distinctive feature of the glycan moiety of phosphatidylinositol anchored membrane proteins, it established the glycosyl-phosphatidylinositol nature of the GP-2 membrane anchor. Since soluble GP-2 is also found in the contents of the granule and is secreted intact into the pancreatic juice, it is likely that one of the mechanisms responsible for its release could be a specific phospholipase. GP-2 is the first glycosyl-phosphatidylinositol-anchored protein that is integral to the membrane of an organelle and not located at the surface of the cell. 0 1988 Academic Press, Inc.
The membrane (1,2)
major is
and
bound
is
common
as
is
present
has
been
strictly of
is
0006-291x/88 Copyright All rights
major
the
contents on
cytoplasmic
paper
in In
in
It
species
(3).
the internal
tail
(6,71.
CRD,
cross-reacting phospholipase trypsin inhibitor; Tris-buffered saline;
granule
an
membrane
protein
integral
side Here
50%
granule
of
of
the
(5).
the we
any
report
the
and
glycoprotein
membrane that
mechanisms
under
immunoreactive The
granule
membrane
proteolytic
secretion
than
of
its
without
basal-level
zymogen
“Elucidation
zymogen
Notwithstanding
more
with
phosphatidyl-
of
determinant of USG; PI-PLC, phosphatidylC; PMSF, phenylaethylsulfonyl fluoride; SDS-PAGE, SDS polyacrylamide gel electrophoUSG, trypanosome variant surface glycoprotein.
$1.50
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
is
pancreatic
intact,
pancreatic
the
a series
secreted
addition, of
the
GP-2.
is
protein
located
the 8th secretion’.
animal
(4). in
any
flbbreviations: inositol-specific SDTI, soybean resis; TBS.,
many
glycoprotein
stimulation
GP-2
*This cellular
the
of
called
to
the
processing
evidence
protein
a glycoprotein
nature,
secretin
distinctive
818
no
Vol.
154,
No.
2, 1988
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Immunoblott.~Ii. Zymiqen granule membranes (150 ~3 proteinf were treated as described in the preceding paragraph with 1 unit of the phosphatidylinositol-specific phospholipase C f roar e, th~gtl~rr@&l~ls , %luble proteins were separated from membrane proteins with Triton X-114, Electrophoretic transfer, renaturat ion, saturation and incubation with 1:500 dilution of antiCRD were done according to Birk EL Koepsell (121, ~mmUr~Oblot5 were developed with lzSI-~radiolabeled anti-rabbit I3 CF(ab’1 2 from donkey3 (FImersham In. 1340)
Otheracrylaeide determined
msmdm: Folyarrylamide gradients according using the standard
gel electrQphoresis to the method of Laenmli Lowry procedure,
819
was
performed an 113). Proteins
6-15X were
Vol.
154,
No.
2, 1988
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
AQUEOUS
DETERGENT
COMMUNICATIONS
. .
A SUPERNATANT
PELLET
B
C
D
A’
AQUEOUS
B’
C’
D’
DETERGENT
. l -‘-..0
1
A
-
0
C
D
A’
B’
C’
D’
0 2
_“” ----,---
A
B
C
D
A’
8’
C’
D’
Figure 1: Digestion of pip zyrogen granule membranes .---with -PI-PLC -.-and 15Opg of pancreatic separation of &hh products bj ultracentrifugation, zymogen granule membrane proteins were incubated for 1 h at 37V in presence of 1.5 units of PI-PLC. The PI-PLC was pretreated for 1 h at 4'C with: lane and al, TDS only; lane b and b', 0.25mTl o-phenanthroline; lane c and cl, 2&i ZnCl,. In lane d and dl, no PI-PLC was added. Rfter incubation the reaction mixture was subjected to centrifugation at 100,OOOg for ih. Proteins recovered in the supernatant (lanes a, b, 3: and d) and pellet (lanes a', b*, cl and d’) were analyzed by SDS-PCIGE. Clrrowheads show molecular mass of standard protein markers of 94, 67, 43, 30, 20 and 14 kDa. Figure 2. Digestion of rat and m zymogen granule membranes with PI-PLC separation of the products into soluble and membrane bound fores using Triton X-114 15Opg of pancreatic zymogen granule membrane proteins from pig -L (top) or rat (bottom) were incubated for 1 h at lb°C in TBS containing 1.2% Triton X-114 in presence of 1.5 units of PI-PLC pretreated as described in Fig 1. After incubation the reaction mixture was subjected to phase separation in Triton X-114 solution (see Wethods). Proteins recovered ia the aqueous (lanes a, b, c and d) and detergent (lanes ai, b*, cl and dl) phases Rrrowheads, see Fig. 1. were analyzed by SDS-PRGE. and
820
a
Vol.
154,
No.
2,
1988
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Anti-CUD
0 A
-0
MEMBRANOUS
SOLUBLE
0 B
E.iQure 5. Iamunoblottinp of PI-PLC solubilized GP-2 with anti-CRD antiserum. Samples consisting of GP-2 from zymogen granule membranes treated with B_, thuringiensis PI-PLC were separated into soluble and membrane bound The detergent fraction before digestion (far right) forms iusing Triton X-114. and soluble fractions after 30 (far left) and 60 min (center) of treatment were subjected to SDS-P1GE, transferred to nitrocellulose and inmunostained Top section fl shows the coomassie with I:500 dilution of anti-CRD antiserum. staining and lower section I3 the anti-CRD imaunoreactivity revealed with C1z"Il anti-rabbit IgG.
821
Vol.
154,
protein
No.
ornithine
membranes
BIOCHEMICAL
2, 1988
in
a
protein5
so
of
far
corcentration function
is
GF-2
7,ymogen
prateirl
consequent-ly
le that
is
surfaces
of has
there antigen
(23)
diffusion is
ttre .an
meRlt?l~arre. important
grari~ile
be
one
attached
to
variant of
the
(21)
antigen
that, i 5 cirni
li~ded
concept
in
three are
the
that
is
nut
i;p-2
a caefficient played 01% xlothec,
ityd~vlysi~
a
GF-2
whether it
15 released
fl-nm
i ri the
pancreatic
juiccie,
the
present
rapid
Thy-l
lateral
lipids,
It
require
3
he released
Sy a pi-~o~pt~olipase
G-2
is
the
for
to
could
been
apical
the
reported very
GF-2 weld
also
a5 clear
iderttical bu
case,
organel-
01. whether &.
high
primary
the
has
well
membranes: to
the is
GF-2
as It
very
intracellular
an
attachment,
COftfel-
time
that this
If
membrar,es in
role
the
by which
ultimtely
these nature
one
the
base-lateral (33) cell -_ ,
membrane,
at
to
its
but
compartment.
the in
that
and
and
Thy-l
to W-2
functional
PI would
in the
I?-
to
ClS>,
and
a membrane
at
via
mci-:artisar ~0irteTit
srith
mobile
pck:ssible
mnb3.iity
pi-otein
acinar
highly
coefficient
has led
be in this
function
, anchorage
tirl?~PfJl-~
iateral
first
pancreatic
to its
(20)
COMMUNICATIONS
Trypanosose
attribilted
granule
to have
a ~secorrdary
due
rleafly
localized
the
known
acetylcholinesterase
would
the
reported
irmunocyto~~hemically protein
proteins proteins.
protein
basic
has been
zn the this
of
RESEARCH
proteins.
such
role
no
the
5urface
117))
ayelin
BIOPHYSICAL
all
cell
wx!
(181,
examples
best
are
way
glycoprateins
surface N-CGM
Cl&),
decarboxylase
such
AND
membranes
C
could
into
high from be
the
Vol.
11. 17L.
154,
No.
2, 1988
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
17.
Holloway, P.W, (1973) final. Biochem. 53, 304-308. Birk, H.-W. and Koepsell, H. (1987) Biochem. Biophys. Res. Commun. 12-22 1 Laesmli, U.K. 119701 Nature 227, 680-685, Little, C. (19811 tiethods Enzyeol. 71, 725-730. Ikezawa, H, and Taguchi, I?. (1881) Hethods Enzymol, 71, 731-741, S.P,, fynther, J. and Clndersson, Sustel in, T, , P&is&, H., Lapinjoki, (1987) Cell 49, 171-176. Lou, H.G. and Cross, G.R.ti. (1985) J. Biol. Chea. Ferguson, H.C\.J.,
18.
14547-14555, Rosenberry,
13. 14, 15. 16,
19. ?@ 21 22. 23.
T , L. ,
Roberts,
M.L.
and
Haas,
R.
(lP86)
Fed.
Proc.
45,
164,
L.C. 260, 2970-
2975. Hestperley, J..J., Edelsan, G.i%. and Cunningham, B.pI. (1987) Proc. Natl. fb:ad. Sci. U.S,FI, 84, 2C107-2011. Cl-tang, P.C., J.C., Fujitaki, J.H., Chiu, K.C. and Smith, R.&b. Y-3, (1986:) Biochemistry 25, 2682-2686. Law, l$,G. and Kincade, P.M. (1985) Nature 318, 63-64. Gem@, H..J., Slot, J.V,, van der Ley, P.I?. and Schef fer , R.C.T. (19811 J. Cell Viol. BP, 653-665. Ishih.ara, A, , Hou, Y, and -!a~obson, K. (1987) Proc, Natl. &ad. Sci. (USR)
84,
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823
