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.

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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.

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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.

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The involvement of calcium in transport of secretory proteins from the endoplasmic reticulum.

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...
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