Cell, Vol. 61, 197-199, April 20, 1990, Copyright 0 1990 by Cell Press
Minireview
The Involvement of Calcium in Transport of Secretory Proteins from the Endoplasmic Reticulum Joseph F. Sambrook
over wide ranges.
Department
that these oscillations
might be involved
of vesicles
correctly
of Biochemistry
University Dallas,
of Texas Southwestern
Medical
School
This review
that carry
discusses folded
the possibility in the formation
secretory
proteins
from the ER to the Golgi apparatus.
Texas 75235-9038
Calcium Ions Are Stored in the Endoplasmic Reticulum The secretory outside
pathway
is poorly
named.
of the cell is not an unbroken
secretory proteins
proteins
make
of membrane-bound specialized
cisterns
organelles,
aged into transport tract
that bind calcium
a series
CRP55 (Koch, 1987). All these proteins contain
clusters
into
aspartate
among hy-
reticu-
drophobic
are
organized
stacks of the Golgi apparatus.
vesicles
proteins
are pack-
that ferry them across the in-
of cytoplasm
to the
next
closed
com-
apparatus
into a series of sepa-
rate cisterns has several advantages: tunity
to establish
different
and maintain
intracellular
competing
secretory
it provides
different
compartments,
biochemical
ed from one another, individual
allows
functions
to be cleanly
and provides
a means
proteins
the discontinuities
serve as checkpoints
into specialized
cistern
that have
traveling The
not been
best
of this
type
of newly synthesized
discriminately
in Gething
in
may in one
of the abundance
during
from
tertiary
are denied
access
proteins
is
1990). Pasin-
and quaternary
confor-
in the ER. Secretory or oligomerize
to transport
vesicles
to the cell surface is blocked in the Golgi appara-
tus or the trans-Golgi By contrast
network.
to the vast amount
about the biochemical of “fixed” organelles, the mechanisms
of information
available
events that occur within the lumen comparatively
by which
ated from the membranes
little is known
transport
vesicles
of donor
organelles
about
are generand how
secretory
proteins
packaged
into them. Work with in vitro systems has shown
that vesicular
that fulfill the appropriate
traffic
requires
between
GTP and
1989). Whereas
GTP remains relatively tle opportunity concentration
criteria
the ER and the Golgi calcium
the intracellular constant
ions
(Beckers
concentration
and therefore
to control the flow of vesicular of cytoplasmic
calcium
provides
could reach 3 mM. ions in the cytoplasm
lower. The resulting
are apand
that
pump
is
electromo-
by the action of
calcium
ions
from
the
fashion. The ER of yeasts
et al., 1989) and mammalian
cells (Burk et al.,
at least one of these pumps, which transfer
ions from the cytoplasm
where they presumably or are loaded
to the lumen of the ER,
bind to phospholipid
into the acidic
head groups
sites of the luminal
calcium
proteins.
Up to 50% of the calcium of mammalian
of inositol
hydrolysis
trisphosphate
of inositol
tol-dependent
ions sequestered
cells can be rapidly
released
[lns(l,4,5PJ]
4,5bisphosphate
phospholipase
in the ER through
the
formed
by
by phosphoinosi-
C (reviewed
in Berridge
and
Irvine, 1989). Until recently, it was believed that lns(1,4,5)P3 was produced
chiefly in the plasma membrane
cloned that encode
since
in the
mglml), the total concentration
in an ATP-dependent
effi-
whose
Al-
up to 30 calcium
of these proteins
of calcium
ATPases
1989) contains
action
of
(Kd=10m5 M), each mole-
can sequester
three orders of magnitude
to external
may
pathway,
have been identified
interspersed
ions in the organelle
The concentration
pro-
and are
Checkpoints
also exist at later stages of the secretory proteins
is and
but only to those
in the ER or degraded.
mutants of secretory
control
is not granted
proteins
their residence
either retained
of calcium
calcium
78), RP60, and
ligand such as calcium.
is not tight
proteins
(Rudolph
secretory
teins that are unable to fold correctly
Balch,
a cationic
ions. Because
vesicles,
for folding
and Sambrook,
to all secretory
that attain a satisfactory
paratus
residues
cule of these
cytoplasm
of quality
sage from the ER to the Golgi apparatus
transport
the binding
binding
example
located (reviewed
ciently
though
to separate
processed
by the ER, where the machinery
oligomerization
mation
of cradling
ion-motive
proteins
BiP(GRP
tracts that could form bends and short helices
capable
segregat-
to the next.
provided
and glutamate
are four proteins
tive force on the ion is counterbalanced
potentially
pathway
secretory
adequately
in
form. In addi-
in the transport
to prevent
an oppor-
conditions
which they can be stored in a concentrated tion,
ions: GRP94,
lumen of the ER (~30-100
partment. Division of the secretory
ions.
Instead,
that
in the lumen of the organelle
of calcium
progress.
On leaving each cistern, the passenger tervening
reservoir
pass through
such as the endoplasmic
lum (ER) and the individual
The ER is the major intracellular Residing
continuous
bound for the cell surface
The route to the
track along which
stimuli.
ma membrane
Recently,
however,
an ER protein homologous
inositol-dependent
is located
in the ER membrane
C (Ben-
for lns(1,4,5)P3 that
and is homologous
to the ryanodine-sensitive
the sarcoplasmic
have been to the plas-
phospholipase
nett et al., 1988), as well as a receptor structure
in response
cDNAs
calcium
in
channel
of
reticulum
(Furuichi
et al., 1989; Mignery
et al., 1989). The possibility
therefore
exists that lns(1,4,5)-
P3 is synthesized also produced
not only in the plasma and consumed
inositol-dependent receptor
phospholipase
may together
membrane
C and the lns(1,4,5)P3
form part of a regulated
nism to drain the ER of part of its calcium of extracellular The fraction sensitive
mecha-
in the absence
signals. of stored
intracellular
calcium
to Ins(1 ,4,5)P3 can be released
by calcium
but is
in the ER. The phospho-
ionophores.
that is not
into the cytoplasm
This result implies that there are
of
at least two pools of calcium ions that are not in open equi-
lit-
librium
traffic, the
ions can oscillate
with one another.
pools are segregated er, for example,
It is not known
whether.these
into distinct compartments
they reflect populations
or wheth-
of calcium
ions
Cell 198
that are bound to different (1,4,5)P3 is generally ling release normal
regarded
of calcium
physiological
ion pump are presumably
ligands within a single compart-
ment such as the ER. Whatever
the arrangement,
Ins-
internal
pools
and indiscriminate
under
A Model
cells can be disturbed
ions in the ER
in a crude way by addition
This treatment
causes
ions to medi-
that draining
the calcium
in this way can cause proteins resident
secreted
(Booth
teins from resident
proteins
ionophores, packaged
More direct evidence of strains
gene encoding
in the secretory
of yeast
a calcium-ATPase
carrying
ions into an intracellular
et al., 1985) but now known 1989), is not essential mal laboratory
trations
in stationary
of calcium,
the anticalmodulin
and are hypersensitive
pmrl mutants efficiently For example, prochymosin,
foreign which
proteins
secreted the pmrl
protein invertase
ed and lacks the outer chain branched through These
added to secretory
tion allows malfolded
suggest
resident
proteins
between
these
two different
one case, the intracellular been drastically
disturbed;
systems
distribution
of calcium
a stimulus
or
and are then
flux
have been observed
cells-usually
such oscillations
there
should
This control
could
not control
in a number of lns(1,4,5)P3
In this case, lns(1,4,5)P3
the ER when the concentration drops
of the second
in the ER concen-
be generated
of calcium
level or when
cytoplasm
of different
in calcium
would
pro-
from the ER.
generation
could be linked to changes
by
ions in the ER
its concentration
below a critical
messenger
and
repetitive
of vesicles
to
in principle
be achieved
For example,
rises to a certain
(Berridge
is no reason
such as the production
in
in response
applied to the plasma membrane 1988). However,
(1,4,5)P3 would
and
from mamyeast and
the conditions proteins
to
in the
threshold.
then would
cause
in concentration
The action of the ion-motive
calcium-ATPase then
return
reducing
and restarting
partment,
is calcium of calcium
to the Of
thread flux.
In
ions has
in the other, mutations
either
in an
of the ER would of the organelle,
of the ion in the cy-
by cycling calcium
are similar
Berridge
to the receptor-controlled by others (see
1989; Meyer and Stryer,
that the concentration
in a constitutive
com-
the cytoplasm.
flux discussed
and Irvine,
1988), which propose
ions between
membrane-bound
or through
models of calcium
P3 oscillates
on the cyto-
the cycle. A similar pattern of oscil-
directly
schemes
fashion.
of lns(1,4,5)-
While this remains
a point of controversy
(Wakui et al., 1989), the model dis-
cussed
only local changes
here requires
concentration
of mam-
The common
the concentration
lation could be achieved
for example,
secretory
of the release
ions to the lumen
toplasm
These
processed
located in the membrane
calcium
thereby
oscillator
residues
of Ins-
ions drain
solic side of the membrane.
In addi-
glycosylat-
Generation
as the calcium
from the ER with an increase
is secreted from
mutant.
be suppressed
the lumen of the ER and another
transport
that loss of PMR7 func-
ionophores.
compartment
channels.
from the ER after treatment
malian cells with calcium
ions flow
into the cytoplasm
Ins(l,4,5)P,-gated
mannose
of the cell. This is reminiscent
either
through
proteins to be released from the ER and transported outside
as waves of calcium
to EGTA and
proteins as they pass
or incompletely
proteins.
ions in the ER fluc-
from the ER to the cytoplasm
the yeast Golgi apparatus. results strongly
and resident
ions to ebb rapidly
mutant in a form that is incompletely
that are normally
manner
in the lu-
and conse-
calcium
for vesicular
from the yeast pm0
efficiently
the calcium
of calcium
many types of mammalian
Generation
However, both foreign proteins are
tion, the yeast secretory
patterns
of calcium,
in the yeast ER do not allow the mammalian to the Golgi apparatus.
lowering
reservoirs
from
high concen-
prolifically
fold into a form that is acceptable
of proteins
to the ER.
Tidal
neighboring
that in wild-type
Presumably,
mem-
by a model based on
of both secretory
in a cyclical
yeasts grow
in the ER of wild-type
very inefficiently.
tuates
tration.
et al.,
such as urokinase
are secreted
target
of the ER membrane
the concentration
membrane
Most significantly,
secrete proteins
Second,
ways.
in the ER.
malian cells, accumulate are secreted
containing
drug trifluoperazine.
cells are trapped
in a
called SSC7 (Smith
low concentrations
cultures
assumptions:
cesses
compartment,
But in its absence,
the following
why
to pump
as PMR7 (Rudolph
ions on the export
ER can be explained
Galione,
when yeasts are grown under nor-
conditions.
poorly on media containing die rapidly
pre-
comes
mutations
storage
such as the ER. The gene, originally
of
pathway.
that is believed
The effects of calcium
returned
that transport
in favor of this hypothesis
In
then de-
the Role of Calcium
into another membrane-bound
pro-
both types of protein into vesicles
them to later compartments
of the ER contents.
to the wrong
the wild-type
from the luminal
calcium
secretory
of the ER. After treatment
cells with calcium become
is
suggests
of intracellular
that segregates
sumably
from studies
which
of the ER, to be rapidly
in the distribution
proteins
to Explain
quent packaging
However,
and Koch, 1989). This finding
that alterations
of the luminal
men causes vesiculation
from the ER
such as GRP94,
in the lumen
can disturb the mechanism
calcium
in concentra-
effects on cellu-
that are not simple to interpret.
normally
of cal-
calcium
um and from the ER. Such drastic changes lar metabolism
to be vesiculation
brane.
both from the extracellular
tion of the ion are likely to have pleiotropic there is evidence
appears packaging
liver their packaged
Draining Calcium Ions from the ER Causes Secretion of Resident Proteins The normal distribution of calcium ions in mammalian cium ionophores.
the concen-
levels. In both
the yeast system at least some of these vesicles
conditions.
flood into the cytoplasm
unable to maintain
ions in the ER at normal
cases, the outcome
as the main valve control-
ions from
tration of calcium
and lns(1,4,5)Ps
stricted to the certain neighboring Modulation
proteins
areas of the ER membrane
of the concentration a mechanism
proteins
and their
of calcium
state. Under
ions within
to retain in the organelle
and newly
that are not folded
port-competent
in both calcium that may be re-
cytoplasm.
the ER provides both resident
utilization
synthesized
and assembled normal
secretory into a trans-
circumstances,
the
ytiireview
matrix of densely packed calcium binding proteins in the ER could form coordination complexes with calcium ions that are bound to the negatively charged phospholipid head groups on the luminal face of the ER membrane, thereby stabilizing the underlying membrane and preventing its vesiculation. According to this model, transfer of calcium ions to the cytoplasm would be the signal that leads to the formation of transport vesicles containing secretory and plasma membrane proteins. The detailed biochemical steps involved in budding of transport vesicles are not known. However, in yeast at least five different genes code for proteins involved in the formation of the small vesicles that mediate transport of secretory proteins from the ER to the Golgi apparatus (Schekman et al., 1990). Genetic and biochemical experiments suggest that some of these proteins are recruited from the cytosol into hetero-oligomeric complexes that are loosely associated with membranes, where they facilitate budding. Perhaps the cyclic assembly and disassembly of these complexes reflect the flux of calcium ions from one side of the ER membrane to the other. At least one major ER protein, BiP, has the ability to bind not only to calcium ions but also specifically to nascent, unfolded secretory proteins. BiR which plays an essential role in the secretory pathway, is thought to stabilize these passenger proteins while they fold and assemble into their three-dimensional conformations (for review, see Pelham, 1989; Gething and Sambrook, 1990). Once assembled, the secretory proteins dissociate from BiP, presumably freeing them to leave the matrix and enter transport vesicles. By contrast, the gel of calcium binding proteins would be largely retained in the lumen of the organelle. Any resident proteins that escaped from the ER could be retrieved in a later compartment by virtue of their C-terminal sequence (KDEL or HDEL) and recycled to the ER (Pelham, 1989). Finally, this model predicts that transport vesicles derived from the ER would normally bud from regions of membrane that not are stabilized by the calcium-protein gel. These regions may be in fixed locations, or they may form in areas of membrane that are temporarily unable to sustain the luminal calcium-protein matrix. Such transitional areas might arise as a consequence of stochastic fluctuations in the distribution or activity of calcium-ATPase, or they may be generated by waves of lns(1,4,5)P3 production by the ER membrane. The artificial evacuation of calcium ions from the lumen that occurs when cells are treated with calcium ionophores would be expected to destabilize the entire calcium-protein gel and its associated membrane. Mass vesiculation and indiscriminate packing of luminal proteins would then follow.
References Beckers, C. J. M., and Balch, W. E (1989). J. Cell Biol. 708, 1245-1256. Bennett, C. F.. Balcarek. J. M., Varrichio, A., and Crooke, S. T. (1988) Nature 334, 268-270. Berridge, M. J., and Galione, A. (1988). FASEB J. 2, 3074-3082. Berridge, M. J., and Irvrne, Ft. F. (1989). Nature 347, 197-205. Booth, C.. and Koch, G. L. E. (1989). Cell 59, 729-737.
Burk, S. E., Lytton. J., MacLennan, Brol. Chem. 264, 18561-18568.
D. H., and Shull, G. E. (1989). J.
Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N.. and Mikoshiba, K. (1989). Nature 342, 32-38. Gething, 65-72.
M. J.. and Sambrook. J. F. (1990). Seminars
in Cell Biol. 7,
Koch, G. L. E. (1987). J. Cell Sci. 8< 491-492. Meyer, T., and Stryer, 5051-5055.
L. (1988). Proc. Natl. Acad.
Sci. USA 85,
Mignery. G. A., Stidhof, T. C., Takei, K., and De Camilli, P (1989). Nature 342, 192-195. Pelham, H. R. B. (1989). Annu. Rev. Ceil Biol. 5, l-23. Rudolph, H. K., Antebi, A., Fink, G. Ft., Buckley, C. M., Dorman, T. E., LeVitre, J., Davidow, L. S., Mao, J., and Moir, D. T. (1989). Cell 58, 133-145. Schekman, R., Baker, D., D’Enfert, C., Hicke, L., Hosobuchi, M., Kaiser, C.. Pryer, N., and Rexach, M. (1990). J. Cell. Biochem. 74C (SuppI.), 14. Smith, R. A., Duncan, 1219-1224.
M. J., and Moor, D. T. (1985). Science
229,
Wakui, M., Potter, B. V. L., and Petersen, 0. H. (1989). Nature 339, 317-320.