Lewin Benjamin (ed.) Genes IX

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The

process

of inserting

into

passing

through

a membrane is

called

protein

translocation.

The

same dilemma

must

be solved

for every

sit-

uation

in which a

protein

passes

through

a mem-

brane. The

protein

presents

a hydrophilic

surface.

but the membrane

hydrophobic.

oil and water,

the two

would

prefer

not to

mix.

The

solution is

to create

a special

structure

in the

membrane

through

which

the

protein

can

pass.

There are three

different

types

of arrangements

for

such structures.

The

endoplasmic

reticulum,

mitochondria,

and

chloroplasts

contain

proteinaceous

struc-

tures embedded

in their

membranes

that

allow

proteins

pass

through

without

contacting

the

surrounding

hydrophobic

lipids.

f l+i"tt{f :ti:i.,i

shows

that a substrate protein

binds

directly to

the

structure, is

transported

by it

to the other

side, and then

is released.

Peroxisomes

also have

such structures

their membranes,

but

the substrate

proteins

not

bind directly

to them. lii.;1;9q1

i."1,"r

shows

that instead they

bind to

carrier

proteins

in the

cytosol, the carrier

protein

transported

through

the channel

into the

peroxisome,

and then

the

substrate

protein

released.

For

transport into

the nucleus,

a much larger

and more complex

structure

employed. This

is the nuclear

pore.

F:tirr.l!lt.

i1,."*

shows

that,

although

the

pore provides

the environment

that allows

a substrate

to enter

(or

to leave)

the

nucleus,

it does not

actually

provide

the appa-

ratus that

binds to the

substrate

proteins

and

moves

them through.

Included

in this

appara-

tus

are carrier

proteins

that

bind to the

sub-

strates and transport

them

through

the

pore

the

other side.

Protein

Translocation

May

Be PosttranslationaI

or CotransLationaI

Proteins

that are imported into

cytoplasmic

organelles are synthesized

on free ribosomes

the cytosot.

Protejns

that are imported

into

the ER-Gotgi

system are synthesized

on ribosomes

that are

associated with

the

ER.

Proteins

associate with membranes

means

specific amino acid

sequences catted

signaI

sequences.

SignaI sequences are most

often

leaders that are

located at the

N-terminus.

N-termjnal

signaI sequences

are usuatly

cleaved off

the

protein

during

the

insertjon

process.

l:tilliiil: :i,i,ii Proteins

enter

the ER or a mitochondrion

by binding

to a transtocon

that transports them across

the membrane.

li{riiiti

i,:.lt."i Proteins are transported

into

peroxisomes

by a carrier

protein

that

binds them

the

cytosot,

passes

with

them through the

membrane channel, and

reteases

them

on the other side.

There are two ways

for a

protein

make its

initial contact with a membrane:

The nascent

protein

may associate with

the translocation

apparatus

while it is still

being synthesized

on the

ribosome. This

is called cotranslational

translocation.

10.3 Protein

Translocation

Mav Be Posttranstational or

Cotranslational 227

:ia:-:i.::.

:i-,.! Proteins enter the nucteus by

passage

through very [arge nuctear

pores.

The transport apparatus

is distinct from the

pore

itself and

jnctudes

components

that carry the

protein

through the

pore.

i:i-i:i::i

.ij.i:, Proteins

synthesized on

free ribosomes rn

the cytosol are djrected

after their

release

to specific des-

tinations bv

short siqnaI

motifs.

The

protein

may be released from a ribo-

some after translation has

been com-

pleted.

Then the completed

protein

diffuses to the appropriate

membrane

and associates with

the translocation

apparatus. This is called

posttransla-

tional

translocation.

CHAPTER

Protein

Localization

The location of

a ribosome depends on

whether

the

protein

under

synthesis is associ-

ating with

a membrane cotranslationally:

Cotranslational

translocation is used for

proteins

that enter the endoplasmic

reticulum

(ER).

The consequence of this

association

that the

ribosome is local-

ized to the surface of

the ER. The ribo-

somes

are associated with the ER

membranes during

synthesis

these

proteins,

and therefore are found in

membrane

fractions

the

cell;

thus

they

are sometimes described as

"membrane

bound."

AII other ribosomes are

located in

the

cytosol; because

they are not associated

with

any organelle and

fractionate

sep-

arately

from membranes, they are some-

times called

"free

ribosomes." The free

ribosomes synthesize

all

proteins

except

those that are translocated cotransla-

tionally.

The

proteins

are released into

the cytosol when their synthesis is com-

pleted.

Some of

these

proteins

remain

free in the cytosol

quasi-soluble

form;

others

associate with macromolecular

cytosolic structures, such as

filaments.

microtubules, centrioles, etc.,

are

transported to the

nucleus,

or associate

with membrane-bound organelles by

posttranslational

translocation.

To associate with a membrane

(or

any other

type of structure), a

protein

requires

an appro-

priate

signal, typically a sequence motif that

causes

to be

recognized

a translocation

sys-

tem

(or

to be assembled

into

a macromolecu-

lar structure).

*Ifillitfl 1*.5 summarizes some signals used

proteins

released from cytosolic ribosomes.

Import into the nucleus results from the

pres-

ence of a variety of

rather short

sequences within

proteins.

These

"nuclear

localization

signals"

enable the

proteins

pass

through nuclear

pores.

One type of signal that determines trans-

port

to the

peroxisome

is a very

short C-terminal

sequence. Mitochondrial and chloroplast

pro-

teins are synthesized on

"free"

ribosomes;

after

their release into the cytosol they associate

with the organelle

membranes

by means of

N-terminal

sequences of

-25

amino

acids in

length that are recognized

receptors

on the

organelle envelope.

Proteins that reside within the reticuloen-

dothelial system enter the ER while

they are

being synthesized. The

principle

of cotrans-

lational translocation is

summarized in

Mitochondrion N-terminal Amphipathichelix 12-30

Chloroplast N-terminal Charged

>25

Nucleus Internal Basic

or bipartite 4-g

Peroxisome

C-terminal Short

peptide

3-4

222

lir,,i,;ii:r.

iir.i;.

An important

feature

of this sys-

tem is that

the nascent

protein

is responsible

for

recognizing

the translocation

apparatus.

This

requires

the signal for

cotranslational

translo-

cation to be

part

of the

protein

that is first

syn-

thesized,

and, in fact,

it is usually

located

at the

N-terminus.

common feature

is found

proteins

that

use N-terminal

sequences

to be

transported

cotranslationally

the ER

posttranslation-

ally

to mitochondria

or chloroplasts.

The

N-terminal sequence

comprises

a leader

that

not

part

of the mature

protein.

The

protein

carrying

this leader is

called a

preprotein

and

a transient

precursor

the mature

protein.

The leader is

cleaved from

the

protein

during

protein

translocation.

l;jiii.iir ir

;,-r.,"1

Proteins can enter the

ER-Gotgi

pathway

on[y

by associating with

the

endoptasmic

reticulum white they

are

being synthesized.

liir,ilillr

:r,

1116roOnobic

regions of

proteins

areintrinsi-

calty

interactive.

and unless

prevented

wit[ aggregate with

one another when a

protein

is synthesized

(or

denatured).

acquisition of

proper

structure

requires the assis-

tance

a chaperone.

Protein folding

takes

place

interactions

between reactive surfaces.

Typically these

sur-

faces

consist of

exposed

hydrophobic side chains.

Their interactions form a

hydrophobic core.

The

intrinsic reactivity of

these surfaces

means that

incorrect interactions

may occur unless the

process

controlled.

;;:!iii.lir.ii:

lri.l' illustrates

what

would

happen. As a newly synthesized

protein

emerges from the

ribosome, any

hydrophobic

patch

in the sequence

is likely to

aggregate with

another hydrophobic

patch.

Such associations

are likely to occur at

random

and therefore will

probably

not represent the

desired conforma-

tion

of the

protein.

Chaperones

are

proteins

that mediate

cor-

rect

assembly by

causing a

target

protein

acquire one

possible

conformation

instead of

others. This is accomplished

by binding

to reac-

tive surfaces in the

Iarget

protein

that

are

exposed during the assembly

process

and

pre-

venting

those

surfaces

from interacting

with

Chaperones

May Be

Required

for Protein

Fotding

Proteins that

can acquire

their conformation

spontaneousty

are said to setf-assemb[e.

Proteins can

often assembte into

atternative

structures.

chaperone directs

protein

into

one

particu[ar

pathway

by excluding

alternative

pathways.

Chaperones

prevent

the formation

jncorrect

structures

interacting

with

unfoLded

proteins

prevent

them

from

fotding incorrectty.

Some

proteins

are able to

acquire their mature

conformation spontaneously.

test for this

ability is to

denature the

protein

and deter-

mine whether it

can then

renature into

the

active form.

This

capacity is

called self-

assembly. A

protein

that

can self-assemble

can

fold

or refold into

the active

state frorn

other

conformations, including

the

condition

which it is initially

synthesized. This implies

that the internal interactions

are intrinsically

directed toward

the right

conformation. The

classic

case

that of ribonuclease;

it was

shown

in the 1970s that,

when the

enzyme is dena-

tured, it can renature

vitrl

irrto

the correct

conformation. More

recently the

process

intrinsic folding has

been described

in detail

for some small

proteins.

When correct folding

does not happen, and

alternative

sets of

interactions

can occur, a

pro-

tein may become trapped in

a stable conforma-

tion that is not the intended

final form. Proteins

in this

category cannot self-assemble.

Their

10.4 Chaperones

May Be Required

for Protein FoLding

223

Chaperones

Are Needed

by Newly Synthesized

and by

Denatured

Proteins

Chaperones act on

newly

synthesized

proteins,

proteins

that are

passing

through

membranes.

oroteins that have been denatured.

Hsp

(heat

shock

protein)

and some associated

proteins

form

major

class

of chaperones

that act

on many target

proteins.

Group

I and

group

II chaperonins are [arge

oligomeric assembties

that act on target

proteins

they sequester

in internal cavities.

Hsp90 is a speciatized chaperone that acts on

proteins

of signaI transduction

pathways.

The ability of chaperones to recognize incorrect

protein

conformations allows them to

play

two

related roles concerned with

protein

structure:

When a

protein

is initially synthesized-

that

to say, when

exits the

ribosome

to enter the cytosol-it appears in an

unfolded

form.

Spontaneous folding

then

occurs

as the emerging

sequence

interacts

with

regions

the

protein

that

were synthesized

previously.

Chaper-

ones

influence the folding

process

controlling

the accessibility

of the

reac-

tive surfaces. This

process

is involved in

initial

acquisition of

the

correct confor-

mation.

When a

protein

denatured, new

regions are exposed and become able to

interact. These interactions

are similar

to those that occur when a

protein (tran-

siently)

misfolds

it is initially

synthe-

sized.

They

are

recognized

by chaperones

as comprising

incorrect

folds. This

process

is involved in recognizing

pro-

tein that has been denatured

and either

assisting renaturation or leading

to its

removal

by degradation.

Chaperones may also

required

to assist

the formation

of oligomeric structures and for

the transport of

proteins

through membranes.

persistent

theme in membrane

passage

is that

control

(or

delay) of

protein

folding is

an impor-

tant feature.

fiG$RE

1fl.S shows

that it may be

necessary to maintain a

protein

in an unfolded

state before it

enters the

membrane

because

the

geometry

passage:

the mature

protein

could simply be too large to fit into

the available

channel. Chaperones may

prevent

protein

f,IG{Jft[

1*.S Chaperones

bind to interactive regions

proteins

as they are synthesized

prevent

random

aggregation. Regions

of the

protein

are

released

interact in

orderly manner to

give

the

proper

conformation.

other regions of the

protein

to form

incorrect

conf

ormation. Chaperones f unction by

prevent-

ing

formation

incorrect

structures

rather

than

promoting

formation

of correct structures.

ffiSUftE 10.8

shows an example in which a chap-

erone in effect sequesters a hydrophobic

patch,

thus allowing interactions

to occur that would not

have

been

possible

in its

presence,

as can be seen

by comparing

the

result with

Figure I0.7.

An incorrect

structure may be formed either

by the misfolding

of a single

protein

or by inter-

actions

with another

protein.

The density of

proteins

in the cytosol is high. and

"macromol-

ecular crowding"

can

increase

the efficiencies

of many reactions

compared to the rates

observed in vitro.

Crowding can cause folding

proteins

to aggregate,

but chaperones can coun-

teract this effect. Thus

one role of chaperones

may be to

protect

protein

so that it can fold

without

being adversely affected

by the crowded

conditions in

the cytosol.

We do not know what

proportion

pro-

teins

can self-assemble, as opposed

to those that

require

assistance from a chaperone.

(It

is not

axiomatic

that a

protein

capable of self-assem-

bly invitro actually

self-assembles invivo,

because

there

may be rate differences

in the two condi-

tions, and chaperones

still could be involved in

vivo.There

is, however,

a distinction to

be drawn

between

proteins

that can in

principle

self-

assemble

and those that in

principle

must have

chaperone to assist in

acquisition of the cor-

rect

structure.)

224

CHAPTER 10

Protein Locatization

Protein

is inserted

into closed chamber

ti:":ijr:{

!r-t.ii A

protein

is constrained

to a narrow

pas-

sage

crosses a membrane.

i:ili:!{i

il} ii;

Proteins

emerge from

the ribosome

from

passage

through a

membrane

an unfolded state that

attracts chaperones

to bind and

protect

them from mjs-

fotdi ng.

from

acquiring a conformation

that would

pre-

vent

passage

through

the membrane; in

this

capacity, their

role

is basically

to maintain the

protein

in an unfolded, flexible

state.

Once the

protein

has

passed

through

the membrane, it

may require another

chaperone to

assist with

folding to its mature

conformation in much

the

same way that a cytosolic

protein

requires assis-

tance from a chaperone

as it emerges from the

ribosome. The

state of the

protein

emerges

from a membrane is

probably

similar to that as

it emerges from the ribosome-f35i6allrz

extended in a more

less

linear condition.

TWo major

types of chaperones have

been

well characterized. They

affect {olding through

two different types of mechanism:

+'iii,liiii' .i

j,:":iL:

shows that the Hsp70

sys-

tem consists of individual

proteins

that bind

to, and act on, the substrates

whose

folding

is to be controlled.Ilrecognizes proteins

*li;Li$?il

!i.:.,i'r

chaperon'in

forms

[arge oligomeric

complex and

fotds

substrate

protein

within its

interior.

they are synthesized

emerge from

membranes

(and

also when

they are

denatured

by stress).

Basically,

it con-

trols the

interactions between

exposed

reactive regions

of the

protein,

enabling

it to

fold into the correct

conformation

in situ. The components

the system

are

Hsp70, Hsp40,

and GrpE.

The name

of the system

reflects

the original

iden-

tification of

Hsp70 as a

protein

induced

heat shock.

The Hsp70 and

Hsp40

proteins

bind

individually

to the sub-

strate

proteins.

They use

hydrolysis of

ATP to

provide

the

energy

for

changing

the structure

of the

substrate

protein

and work

in conjunction

with an

exchange

factor that

regenerates

ATP

from ADP.

li{:fili:

lir'..1 I shows thatachaperoninsys-

tem clnsists

of a large

oligomeric assembly

(represented

a cylinder).

This assembly

forms

structure

into which

unfolded

proteins

are

inserted.

The

protected

en-

vironment directs

their

folding. There

are

two types

of chaperonin

system:

GroEL/GroES

is found

in all classes of

organism

and TRiC

is found

in eukary-

otic

cytosol.

The components

the systems

are sum-

marized

iitiii.ii;tt

.i{i.

tii. The Hsp70

system and

the

two

chaperonin

systems

all act on

many

different substrate

proteins.

Another

system,

the Hsp90

protein, functions

in conjunction

10.5 Chaperones

Are

Needed by

NewLy Synthesized

and by

Denatured

Proteins 225

System Structure/function

Individual chaoerones

Hsp70

system

Hsp70

(DnaK)

ATPase

Hsp40

(DnaJ)

Stimulates ATPase

GrpE

(GrpE)

Nucleotide

exchange factor

Hsp90

Functions on

proteins

involved in

sional transductior

Oligomeric structures

(chaperonins)

Group I

Hsp60

(GroEL)

Forms two heptameric rings;

Hsp10 (GroES)

Forms

cap

Group ll

TR|C

Forms

two octameric rings

FIGURE

10.12 Chaperone famities have

eukaryotic and

bacteriaI counterparts

(named

parentheses).

tein" is that increase

temperature causes

pro-

duction of heat shock

proteins

whose function

is to minimize the damage caused to

proteins

heat

denaturation.

Many of the heat

shock

pro-

teins are chaperones and were first discovered,

and named, as

part

of the heat shock response.)

The Hsp70 Family

Is Ubiquitous

Members of the Hsp70 fami[y are found in

the

cytosot, in the ER. and in mitochondria

and

chtorootasts.

Hsp70 is a chaperone that functions

on target

proteins

in conjunction with DnaJ

and GrpE.

The Hsp70 family is found in

bacteria, eukary-

otic cytosol, in the

ER,

and in chloroplasts and

mitochondria. A typical Hsp70 has two

domains:

the

N-terminal domain

ATPase

and the C-

terminal domain binds the substrate

polypep-

tide. When

bound

to ATP, Hsp70

binds and

releases substrates rapidly; when

bound to ADP,

the reactions

are slow.

Recycling

between these

states

is regulated

by two other

proteins,

Hsp40

(DnaJ)

and GrpE.

FIGURE

10.13 shows that Hsp40

(DnaJ)

binds

first to a nascent

protein

emerges from

the

ribosome. Hsp40 contains a region

called the J

domain

(named

for DnaJ), which interacts

with

Hsp70. Hsp70

(DnaK)

binds to both Hsp40 and

to the

unfolded

protein.

effect, two interact-

ing chaperones bind to the

protein.

The

J domain

accounts

for

the specificity of the

pairwise

inter-

action

and drives a

particular

Hsp40 to

select

the appropriate

pafiner

from

the Hsp70 family.

The interaction of Hsp70

(DnaI()

withHsp40

(DnaJ)

stimulates the ATPase

activity of Hsp70.

The ADP-bound

form of the complex

remains

associated with the

protein

substrate

until GrpE

displaces the ADP. This causes loss

of Hsp40 fol-

lowed by dissociation of Hsp70.

The Hsp70

binds

another ATP and the

rycle

can be repeated.

GrpE

(or

its equivalent) is found

only in

bacteria,

mitochondria,

and chloroplasts;

in other loca-

tions, the

dissociation

reaction

coupled to ATP

hydrolysis

in a more complex

way.

Protein

folding is accomplished

by multi-

ple

cycles of association and

dissociation. As

the

protein

chain lengthens,

Hsp70

(DnaK)

may

dissociate from one binding

site and

then

reassociate

with another, thus releasing

parts

of the substrate

protein

to fold

correctly in an

ordered manner. Finally,

the intact

protein

Hsp70

Hsp70-ATP

binds Hsp40

binds

Hsp40 + substrate

substrate

GrpE

Hsp40

is released

Hsp70

dissociates

disolaces ADP

y,.

(gr

()r

FIGURT

10.13

Hsp40

binds the

substrate and then Hsp70. ATP hydrolysis

drives

conformationaI

change. GrpE

displaces the ADP;

this causes the chaperones to

be released.

Muttipte

cycles

of association and dissociation

may occur during

the fotding

of a

substrate

protein.

with Hsp70.

but

directed against

specific classes

proteins

that are involved in

signal transduc-

tion, especially

the steroid hormone

receptors

and signaling

kinases. The Hsp90

system's basic

lunction

is to maintain its

targets in an

appro-

priate

conformation

until they

are stabilized by

interacting

with

other components

of the

pathway. (The

reason

many of these

proteins

are

named

"Hsp,"

which stands for

"heat

shockpro-

CHAPTER 10 Protein

Locatization

226

released from

the ribosome

folded

into its

mature

conformation.

Different

members

of the Hsp70

class func-

tion on various types

of target

proteins.

Cytoso-

Iic

proteins (the

eponymous

Hsp70

and a related

protein

called Hsc70)

act

on nascent

proteins

ribosomes.

Variants

in the ER

(called

BiP

Grp78 in higher

eukaryotes

and I(ar2 in

Saccha-

rlmyces cerevisiae),

or in

mitochondria

or chloro-

plasts,

function

rather

similar manner

proteins

as they

emerge into

the interior

of the

organelle on

passing

through

the membrane.

What

feature does

Hsp70 recognize

in a tar-

get protein?

binds to a linear

stretch

amino

acids

embedded in a hydrophobic

context. This

precisely

the sort

motif

that is

buried in the

hydrophobic

core

of a

properly

folded, mature

protein.

Its

exposure

therefore indicates

that

the

protein

is nascent

or denatured.

Motifs

this nature

occur about every

forty amino

acids.

Binding to

the motif

prevents

it from misaggre-

gating

with another

one.

This mode

of action explains

how the Hsp70

protein

Bip can fulfill

two functions:

to assist in

oligomerization

and/or folding

of newly

translo-

cated

proteins

in the ER, and

to remove misfolded

proteins.

Suppose that BiP

recognizes

certain

pep-

tide sequences that

are inaccessible

within the

conformation

of a mature,

properly

folded

pro-

tein. These

sequences are exposed

and attract BiP

when the

protein

enters

the ER lumen in

an essen-

tially one-dimensional

form. If

protein

is mis-

folded

or denatured, it may

become exposed

its

surface instead of being

properly

buried.

SignaL Sequences

Initiate

Trans[ocation

Proteins

associate

with

the ER

svstem ontv

cotra

s[ationat[y.

The signal sequence

of the substrate

protein

responsibte for membrane

association.

Proteins

that associate

with membranes

via

N-terminal Ieaders

use a hierarchy

of signals to

find

their

final

destination. In

the case of the

reticuloendothelial system,

the ultimate location

of a

protein

depends

on how it is

directed as it

transits the

endoplasmic reticulum

and Golgi

apparatus. The leader

sequence itself introduces

the

protein

to the membrane;

the intrinsic

con-

sequence of the interaction

is for the

protein

pass

through

the

membrane

into

the compart-

ment on the other

side. For a

orotein

to reside

!r.ii"lii1;;|'

i,:.

':,

Proteins that

enter the ER-Gotgi

pathway

may

flow through to the

ptasma

membrane

may be

diverted to other destinations

by specific signa[s.

within the membrane,

a further signal

required to stop

passage

through the

membrane.

Other types

of signals

are

required for a

protein

to be sorted to a

particular

destination,

that is,

to remain within the

membrane or

lumen of

some

particular

compartment.

The

general

process

finding its ultimate

destination by

transport through successive

membrane sys-

tems is called

protein

sorting

or targeting,

and is discussed in

protein

trafficking.

The

overall

nature of the

pathway

sum-

marized

!,',i;iii

ii',..1rr. The

"defaultpathway"

takes a

protein

through

the

ER, into the Golgi

apparatus, and on to the

plasma membrane.

Proteins that

reside in the

possess

C-terminal

tetrapeptide

(KDEL,

which

actually

provides

a signal

for them

to return to the

from

the Golgi apparatus).

The signal that diverts

protein

to the lysosome

is a covalent

modifi-

cation: the addition

of a

particular

sugar

residue.

Other

signals

are required

for a

protein

become a

permanent

constituent

of the Golgi

apparatus or the

plasma

membrane.

There

is a

common starting

point

for

proteins

that asso-

ciate with,

pass

through.

the reticuloendothe-

lial system 0f

membranes.

These

proteins

can

associate with the membrane

only

while they

are

being synthesized.

The

ribosomes synthesizing

these

proteins

become

associated

with

the ER,

enabling the nascent

protein to be cotransla-

tionally transferred to

the membrane.

Regions

in which ribosomes are

associated

with the

are sometimes

called the

"rough

ER," in contrast

with the

"smooth

ER"

regions that

lack associ-

ated

polysomes

and which

have

a tubular

rather

10.7 SignaI Sequences

Initiate

Translocation 227

than sheetlike appearance. i-:ilijiii; li:i.:i: shows

ribosomes in

the

act

transferring nascent

pro-

teins

membranes.

The

proteins

synthesized at the rough ER

pass

from the ribosome directly to the mem-

brane.

Next they are transferred to the Golgi

apparatus,

and finally they are directed to their

ultimate destination,

such as the

lysosome

secretory

vesicle

plasma

membrane. The

process

occurs within a membranous environ-

ment as the

proteins

are

carried between

organelles in

small

membrane-coated

vesicles.

Cotranslational

insertion is directed by a sig-

nal sequence. Most

often this is a cleavable

leader

sequence of l5 to 30

N-terminal amino

acids. At

or close

to the

N-terminus are several

polar

residues,

and

within

the

Ieader is

hydrophobic

core consisting exclusively or

very largely

of hydrophobic amino acids. There

no other

conservation of sequence.

iii--i=iF:i

.iL:.:r:

gives

an example.

The signal

sequence

both

necessary

and

sufficient to

sponsor transfer of any attached

polypeptide

into the target membrane. A

sig-

nal

sequence added to

the N-terminus of a

glo-

i:+***i i

*..1 r

The

endoplasmic reticutum

consists of a

highty fotded

sheet

membranes

that extends from the

nucteus. The

sma[[ objects attached to

the outer surface of

the membranes

are

ribosomes.

Photo courtesy

Lelio

Orci.

University

of Geneva, Switzertand.

bin

protein,

for

example,

causes it to

be secreted

through cellular membranes

instead

of remain-

ing in the cytosol.

The signal sequence

provides

the connection

that enables the

ribosomes

to attach to the mem-

brane. There is no intrinsic difference between

free ribosomes

(synthesizing

proteins

the

cytosol) and ribosomes that are attached to the

ER. A

ribosome

starts synthesis of a

protein

with-

out knowing whether the

protein

will be synthe-

sized in the

qtosol

or transferred to a membrane.

It is the synthesis of a signal sequence that

causes

the ribosome to associate with a membrane.

The

SignaI

Sequence

Interacts

with the SRP

The

signal sequence

binds to the

SRP

(signat

recognition

particte).

Signa[-SRP binding causes

protein

synthesis to

pause.

Protein synthesis resumes when the SRP b'inds

the SRP receptor in the membrane.

The

signal sequence

cleaved

from

the

translocating

protein

by the signal

peptidase

located on the

"inside"

face

of the membrane.

Protein translocation can

be divided into two

general

stages:

first, ribosomes

carrying nascent

polypeptides

associate with the membranes,

and then the nascent chain is

transferred to the

channel and translocates through it.

The attachment of ribosomes to membranes

requires the signal recognition

particle

(SRP).

The

SRP has two important abilities:

can bind to the signal sequence

nascent

secretory

protein.

can bind to a

protein (the

SRP recep-

tor) located in

the membrane.

The SRP and SRP receptor function

catalyt-

ically

to transfer a

ribosome

carrying

nascent

f l+Uf

it.]"

i! ti The signaI

sequence of bovine

growth

hormone

consists of the N-termina[ 29

amino

acjds and has a central highty

hydrophobic region,

preceded

flanked

by regions containing

polar

amino

acids.

Protein

Locatization

Initiation

{ Polar}<(- Hydrophobic

core +

228

CHAPTER 1O

protein

to the membrane. The

first

step

the

recognition

the

signal sequence

by the

SRP.

The

SRP then binds to the

SRP receptor

and the

ribosome

binds to the membrane.

The stages

translation

of membrane

proteins

are

summa-

rized

ii.l'l;i

r:r

The role of

the SRP receptor in

protein

translocation is transient.

When the

SRP binds

to the

signal sequence, it arrests

translation.

This usually happens

when

-70

amino acids

have

been incorporated into

the

polypeptide

chain

(at

this

point

the

25-residue

leader has

become exposed,

with the next

-40

amino acids

still

buried

the ribosome).

When the

SRP binds to the

SRP

receptor,

the

SRP releases the signal

sequence. The ribosome

becomes

bound by another component

of the

membrane. At

this

point,

translation

can resume.

When the ribosome has

been

passed

on to the

membrane, the

combined role

of SRP and SRP

receptor

has been

played.

They

now recycle and

are free to

sponsor the association

of another

nascent

polypeptide

with the

membrane.

This

process

may

needed

to control the

conformation

of the

protein.

If the nascent

pro-

tein were released into

the cytoplasm, it could

take up a conformation in

which it might be

unable to traverse the

membrane. The ability

the SRP to inhibit

translation while the ribosome

being

handed

over to the membrane is

there-

fore important

preventing

the

protein

from

being released into

the aqueous environment.

The signal

peptide

cleaved from a translo-

cating

protein

by a complex

five

proteins

called the signal

peptidase.

The

complex is

several times more abundant

than the SRP and

SRP

receptor. Its

amount is equivalent roughly

to the amount of bound ribosomes,

suggesting

that

functions in a structural

capacity.

It is

located on the lumenal face

of the ER mem-

brane, which implies

that the entire signal

sequence must

cross the

membrane

before cleav-

age occurs.

Homologous

signal

peptidases

can

be recognized in eubacteria,

archaea, and

eukaryotes.

The

SRP

Interacts

with

the SRP Receptor

The SRP is a comptex of 75 RNA with

six

proteins.

The bacterial equivatent to the

SRP

a complex of

4.55 RNA with two

proteins.

The

SRP

receotor is

a dimer.

GTP

hydrotysis releases

the SRP

from

the SRP

receotor

after their

interaction.

The interaction between the

SRP and the SRP

receptor is the key event

in eukaryotic

transla-

tion in transferring a

ribosome carrying

a nas-

cent

protein

to the

membrane.

An analogous

interacting system exists

in bacteria,

although

its role is more restricted.

The

SRP

is an I I S ribonucleoprotein

com-

plex,

containing six

proteins

(total

mass 240 kD)

and a small

(305

base,

I00 kD)

75 RNA.

rl{iiil'i ;i: ii,

shows that

the

75 RNA

provides

the structural backbone

of the

particle;

the

indi-

vidual

proteins

not assemble

in its absence.

The 75 RNA of the

SRP

pafiicle

is divided

into

two

parts.

The 100 bases

at the

5'end and

the 45 bases at the 3'end

are closely

related to

ii.lilii:r

1, I

Ribosomes

synthesizing

secretory

pro-

teins are attached to

the membrane

via the

signal sequence

the

nascent

potypeptide.

10.9

The

SRP

Interacts

with the

SRP Receptor

+ Alu RNA domain

.++

RNA

domain *

Signal sequence

emerges from ribosome;

SRP approaches in

extended

form

binds

stgnal sequence

and

bends at hinge

io contact ribosome

Signal

sequence

tI6URt 10.18

75 RNA ofthe

SRP

has

two domains. Proteins

bind as shown

on the two-dimensionaI

diagram above to

form

the crystal

structure shown betow. Each function

the SRP

associated with

a discrete

oart of the structure.

the sequence

of Alu RNA,

a common mam-

malian

small RNA. They

therefore define

the

Alu

domain. The remaining part

the RNA

comprises

the

S domain.

Different

parts

of the

SRP structure depicted

in Figure

10. l8 have

separate functions

pro-

tein

targeting.

SRP54 is the most important

sub-

unit. It is located

at one end of the

RNA structure

and

is directly responsible

for recognizing

the

substrate

protein

binding to the

signal

sequence.

It also

binds to the

SRP receptor in

conjunction

with the

SRP68-SRP72

dimer that

is located

at the central

region

of the

RNA.

The

SRP9-SRPl4

dimer is located

the other end

of the molecule;

it is responsible

for

elongation

arrest.

The

SRP

a flexible

structure. In its

unen-

gaged

form

(not

bound to signal

sequence), it

quite

extended,

as can be

seen from the

crys-

tal

structure

Figure

10. I 8. FIGURT

10.19

shows

that

binding

to a signal

sequence triggers

change

conformation. The protein

bends at

a hinge

to allow the

SRP54 end

to contact the

ribosome

the

protein

exit site,

while the

SRPI9

swings around

to contact

the ribosome

at the elongation

factor

binding site.

This enables

to cause

the elongation

arrest that

gives

time

Protein

Localization

FIGURI 1*"19

SRP binds to a signal

sequence as it

emerges from

the

ribosome.

The binding

causes the SRP

to change conformation by bending

at a

"hinge,"

altow-

ing

SRP54 to contact the ribosome at the

protein

exit site

white

SRP19

makes

a second set of contacts.

for

targeting to the translocation

site

on the

membrane.

The

SRP

receptor

is a dimer

containing sub-

unirs

SRcx,

(72

kD) and SRp

(30

kD).

The

sub-

unit is

an integral membrane

protein.

The

amino-terminal

end of the large u

subunit is

anchored

by the

subunit. The

bulk of the

pro-

tein

protrudes

into

the cytosol. A large

part

the

sequence of the cytoplasmic

region

of the

protein

resembles

a nucleic acid-binding pro-

tein with many

positive

residues.

This suggests

the

possibility

that the

SRP

receptor

recognizes

the 75 RNA in

the SRP.

There is a counterpart

to SRP in

bacteria,

although it

contains fewer comporLerrts.

coli

contains a 4.5S RNA

that associates

with ribo-

somes and is

homologous to

the 75 RNA

of the

SRP. It

associates

with

two

proteins:

Ffh is

homologous

SRP54 and FtsY is

homologous

to the

cr subunit of the

SRP receptor.

In fact,

FtsY

replaces the functions

both the or

and

SRP subunits; its

N-terminal domain

substitutes

for

SRPp in membrane

targeting,

and the

230

CHAPTER