Lewin Benjamin (ed.) Genes IX

The leader sequence

and the

transported

protein

represent

domains

that fold indepen-

dently. Irrespective

of the sequence

to which it

is attached, the leader

must

be able to fold into

an appropriate structure

to be recognized

by

receptors on the

organelle envelope.

The

attached

polypeptide

sequence

plays

no

part

in

recognition of the

envelope.

What restrictions

are there on transporting

a hydrophilic

protein

through

the hydropho-

bic membrane? An insight

into this

question

is

given

by the observation

that methotrexate,

a

ligand for

the enzyme DHFR,

blocks transport

into mitochondria

of

DHFR

fused to a mito-

chondrial

leader.

The tight binding

of methotrex-

ate

prevents

the

enzyme from unfolding

when

it is

translocated through the membrane.

The

sequence of the transported

protein

is irrele-

vant for targeting

purposes;

however, in

order

to follow its leader through

the membrane, the

protein

requires

the flexibility

to assume an

unfolded conf ormation.

Hydrolysis of ATP is required

both outside

and inside for translocation

across the membrane.

It

may be

involved

with

pushing

the

protein

from

outside and

pulling

from

inside. In the cases

of

mitochondrial

import

and bacterial

export, there

is also a requirement for

an electrochemical

potential

across

the

inner

membrane to transfer

the amino terminal

part

of the leader.

imported into a mitochondrion

is

to

move

through both

membranes

into the matrix.

This

property

is conferred

by the N-terminal

part

of

the

leader

sequence.

A

protein

that

is localized

within the

intermembrane space

or in the

inner

membrane itself

requires an additional

signal,

which specifies

its destination

within

the

organelle. A multipart

leader contains

signals

that function

in

a

hierarchical

manner, as

sum-

marized in

iri{-;,,iFil

1fi"}$. The

first

part

of the

leader

targets the

protein

to the organelle,

and

the second

part

is required

if its destination

is

elsewhere than the

matrix.

The two

parts

of

the

leader

are

removed by successive

cleavages.

Cytochrome cl

is an example.

It is bound

to

the inner

membrane and

faces the

intermem-

brane space. Its

leader sequence

consists of

sixty-

one amino

acids and can

be divided

into regions

with different

functions.

The sequence

of the first

thirty-two amino

acids alone,

or even

the

N-terminal

half

of

this

region, can transport

DIIFR

all the way into the

matrix.

Thus the

first

part

of

the leader sequence

(thirty-two

N-terminal

amino

acids) comprises

a

matrix-targeting

signal.

The

lnner membrane

F:td;tJi;1{

j

#" }*,

Mitochondria

have

receptors

for

protein

transport

in the outer

and

inner

membranes.

Recogni-

tion at the outer

membrane

may lead

to transport

through

both

receptors

into the

matrix.

where the

leader

is

cleaved.

If

jt

has a

membrane-targeting

signa[,

it may

be

reexported.

10.17 A Hierarchy

of Sequences

Determines

Location

within

0rganeltes

247

@

A Hierarchy

of Sequences

Determines

Location

within

0rganelles

The N-terminal

part

of a leader sequence targets a

protein

to

the mitochondriaI

matrix or chloroptast

[umen.

An

adjacent sequence can control further targeting

to a membrane or the intermembrane soaces.

The

sequences are cteaved successivety from the

orotein.

The mitochondrion is

surrounded by an

envelope consisting of two membranes. Pro-

teins imported into mitochondria may be located

in

the outer

membrane,

the intermembrane

space, the

inner

membrane, or the matrix. A

protein

that

is

a component of one of the mem-

branes may be oriented so that it faces one side

or the other.

What is responsible for directing a mito-

chondrial

protein

to the appropriate compart-

ment? The

"default"

pathway

for a

protein

lnitiation

I

|

<- charged leader

region tor mitochondrial targeting

-f>

l++++

Ser

9@ev

Cleavage 1

Continuous uncharged leader region targets inner

membrane

itnt

'lla;

Cleavage 2

'

FIfi:JRi

i*.31

The leader

ofyeast cytochrome c1

contains an

N-terminaI

region that targets

the

pro-

tein

to the

mitochondrion,

followed

by a region that targets

the

(cteaved) protein

to the inner mem-

brane.

The

leader is removed

by two cteavage events.

intact leader,

however,

transports an

attached

sequence-such

as murine DIIFR-into

the inter-

membrane

space.

What

prevents

the

protein

from

proceeding

past

the intermembrane

space when it has

an

intact leader?

The region

following

the matrix-

targeting

signal

(comprising

nineteen

amino acids

of the leader)

provides

another

signal that local-

izes the

protein

at the inner

membrane

or within

the intermembrane

space. For working

purposes,

we

call this the

membrane-targeting

signal.

The

two

parts

of a

leader

that

contains both

tlpes

of signal have

different

compositions. As

indicated

in

Fi*L:Hf

':*.

j

l,

the

thirty-five

N-terminal

amino acids resemble

other organelle

leader

sequences in

the high content

of uncharged

amino acids,

punctuated

by basic

amino acids.

The next

nineteen

amino acids,

however,

com-

prise

an unintemrpted

stretch of r.ncharged

amino

acids

that is long

enough

to span a lipid

bilayer.

This

membrane-targeting

signal resembles

the

sequences

that are involved

in

protein

transloca-

tion into

membranes

of

the ER

(see

Section 10.7,

Signal

Sequences Initiate

Translocation).

Cleavage

of the matrix-targeting

signal is

the sole

processing

event required

for

proteins

that

reside in

the matrix.

This signal

must also

be cleaved from

proteins

that reside in

the inter-

membrane

space; however,

following

this cleav-

Protein

Locatization

age, the membrane-targeting

signal

(which

is

now the

N-terminal sequence of the

protein)

directs the

protein

to

its

destination in

the outer

membrane, intermembrane

space, or inner

membrane.

The signal

then,

in

turn, is

cleaved.

The

N-terminal matrix-targeting

signal func-

tions in

the same manner for

all mitochondrial

proteins.

Its recognition

by a receptor

on the

outer membrane leads

to transport

through the

two membranes.

Note that the

same

protease

is involved in

cleaving the matrix-targeting

sig-

nal, irrespective

of the final

destination

of the

protein.

This

protease

is

a water soluble,

Mg2+-

dependent

enzyme that is located

in the

matrix.

Thus

the N-terminal

sequence must reach

the

matrix, even if

the

protein

ultimately will

reside

in

the intermembrane

space.

Residence

in the matrix

occurs

in the

absence

of any other signal.

If there is

a mem-

brane-targeting

signal, however,

it is

activated

by cleavage of the matrix-targeting

signal. The

remaining

part

of the leader

(which

is now

N-terminal) then

causes the

protein

to

take up

its final

destination.

The nature

of the membrane-targeting

sig-

nal is

controversial.

One model holds

that

tne

entire

protein

enters the matrix,

after

which

the membrane-targeting

signal causes

it to be

reexported

into or

through the inner

mem-

242

CHAPTER

10

brane. An alternative

model

proposes

that the

membrane-targeting

sequence simply

prevents

the rest

of the

protein

from following

the leader

through

the

inner

membrane into

the matrix.

Whichever model

applies,

another

protease

(located

within the intermembrane

space)

com-

pletes

the removal

of

leader

sequences.

Passage

through chloroplast

membranes is

achieved in a

similar manner.

5li:lgS#

t*.*{; illus-

trates the variety of locations

for chloroplast

proteins.

They

pass

the outer and inner mem-

branes of the envelope into the

stroma, a

process

involving the

same types of

passage

as

into

the

mitochondrial

matrix. Some

proteins

are trans-

ported yet

further,

though, across

the stacks of

the thylakoid membrane into

the lumen. Pro-

teins destined for the thylakoid

membrane or

Iumen must cross the

stroma en route.

Chloroplast targeting

signals resemble mito-

chondrial targeting signals. The leader

consists

of

-50

amino acids, and the

N-terminal

half is

needed to recognize the

chloroplast envelope.

A cleavage between

positions

20 and 25 occurs

during or

following

passage

across the

enve-

lope, and

proteins

destined for

the thylakoid

membrane

or

lumen

have a new

N-terminal

Ieader that

guides

recognition

of the thylakoid

membrane. There

are several

(at

least four) dif-

ferent

systems

in

the chloroplast that catalyze

import of

proteins

into the thylakoid membrane.

The

general principle governing protein

transport into mitochondria

and chloroplasts,

therefore,

is

that the N-terminal

part

of the

Ieader targets a

protein

to the

organelle

matrix,

and an additional sequence

(within

the

leader)

is needed

to

localize

the

protein

at the outer

membrane, intermembrane

space, or

inner

membrane.

@

Inner

and 0uter

MitochondriaL

Mem

branes

Have Different

TransLocons

.

Transport through the outer and inner

mitochondriaI membranes uses different

receptor complexes.

.

The TOM

(outer

membrane)

comptex is a large

comptex

in which

substrate

proteins

are directed

to the

Tom40

channel by one of two subcomptexes.

.

Different

TIM

(inner

membrane)

complexes are

used depending

on whether

the substrate

protein

is

targeted

to the inner membrane

or to the

lumen.

.

Proteins

pass

directly from

the TOM to the TIM

comDtex.

Fi*#flil

11.;.:i;t: A

protein

approaches

the chloroptast

from the

cytosoI with a

-50

residue leader.

The

N-terminaI

hatf

of

the leader

sponsors

passage

into the envetope

or

through

it into the stroma.

Cleavage occurs during

envelope

passage.

lT{,lJSl

1iJ":,!j

T0M

proteins

form receptor

comp[ex(es)

that are

needed for transtocation

across the

mitochondr-

iaI outer

membrane.

There are different

receptors

for transport

through each

membrane

in the chloroplast

and

mitochondrion.

In the

chloroplast

they

are called

TOC

and

TIC, and

in the

mitochondrion

they

are

called TOM

and

TIM, referring

to

the outer

and

inner membranes,

respectively.

The TOM complex

consists

of

-9

proteins,

many

of

which are

integral

membrane

proteins.

A

general

model

for the complex

is shown

in

f].ilLlq{

;i}.;,ti}.

The

TOM

aggregate

has

a size of

>500

kD, with

a diameter

of

-138

A, and

forms

an

ion-conducting

channel.

A complex

con-

tains

2 to I

individual

rings

of diameter

75 A,

each

with a

pore

of

diameter

20 A.

Tom40 is deeply

imbedded

in the

mem-

brane and

provides the channel

for transloca-

tion.

It

contacts

preproteins as

they

pass

through

the outer membrane.

It binds

to three

smaller

10.18 Inner and

Outer

Mitochondria[

Membranes

HaveDifferent

Transtocons

243

FIGURE

10.40

Tim

proteins

form

the

comptex

for

transto-

cation

across the mitochondriaI

inner membrane.

proteins.

Tom5,

-6,

and

-7,

which

may be com-

ponents

of the channel

or assembly factors.

There

are two subcomplexes

that

provide

sur-

face receptors.

Tom20

and Tom22

form a sub-

complex with

exposed

domains in the

cytosol.

Most

proteins

that are imported

into mitochon-

dria

are recognized

by the Tom20,22

subcom-

plex,

which

is the

primary

receptor

and

recognizes

the

N-terminal

sequence of the

translocating

protein.

Tom37,70,71 provides

a

receptor for

a smaller

number

of

proteins

that

have internal

targeting

sequences.

When

a

protein

is

translocated

through the

TOM

complex,

it

passes

from

a state in

which

it

is exposed

to the

cytosol into

a state in which

it is

exposed

to the intermembrane

space.

It is

not,

however,

usually released,

but instead

is

transferred

directly to

the TIM

complex. It is

possible

to

trap intermediates

in which

the leader

is

cleaved

by the matrix

protease,

leaving

a major

part

of the

precursor

exposed

on

the cytosolic

surface

of the envelope.

This

suggests that

a

protein

spans the

two membranes

during

pas-

sage. The

TOM

and TIM

complexes

do

not

appear

to interact

directly

(or

at least

do not

form

a detectable

stable

complex),

and they

may therefore

be

linked

simply by a

protein

in

transit.

When

a translocating protein

reaches

the intermembrane

space,

the

exposed residues

may immediately

bind

to a TIM

complex,

whereas

the

rest of the

protein

continues

to

translocate

through

the TOM

complex.

There

are two TIM

complexes

in the inner

membrane.

The TimlT-23

complex

translocates

proteins

to the lumen.

Substrates are recog-

nized by their

possession

of a

positively

charged

N-terminal signal.

Transmembrane proteins

Timl7 through Tim23 comprise

the channel.

FIGURE 10-40

shows that they are associated

with

Ttm44 on the matrix

side of the membrane. Tim

44 in

turn binds the chaperone Hsp70.

This is

also associated with another chaperone,

Mge,

the counterpart to

bacterial GrpE. This

associ-

ation

ensures that when the imported

protein

reaches the matrix, it is

bound by the Hsp70

chaperone. The high

affinity of Hsp70

for the

unfolded

conformation of the

protein

as it

emerges from the inner membrane

helps

to

"pull"

the

protein

through the channel.

A major chaperone

activity in the mito-

chondrial matrix

is

provided

by Hsp60

(which

forms

the same sort of

structure as its counter-

part

GroEL). Association

with Hsp60 is

neces-

sary for

joining

of the subunits

of imported

proteins

that form oligomeric

complexes.

An

imported

protein

may be

"passed

on" from

Hsp70

to Hsp60 in the

process

of acquiring

its

proper

conformation.

The Tim22-54

complex translocates pro-

teins

that

reside

in the inner

membrane.

How

does a translocating

protein

finds its

way

from

the TOM complex

to the appropriate

TIM complex? TWo

protein

complexes

in

the

intermembrane

space escort a translocating pro-

tein from TIM

to TOM. The Tim9-

l0 and Tim8-

I3

complexes act as escorts for

different

sets of

substrate

proteins.

Tim9- l0 may

direct its

sub-

strates to either Tim22-54

orTtm23-17

,

whereas

Tim8-ll

directs substrates only

to Tim22-54.

Some

substrates do not

use either Timg-I0

or

Tim8-13,

so other

pathways

must also

exist.

The

pathways

are summarized

in FIGURH 1Q"41.

What is the role

of the escorting

complexes?

They may

be needed to help

the

protein

exit

from the TOM

complex as well

as for recogniz-

ing the TIM

complex.

FIGURT

10"42

shows

that

a translocating

protein

may

pass

directly

from

the

TOM

channel

to the Tim9,l0

complex,

and

then into the Tim22-54

channel.

A mitochondrial protein

folds

under

differ-

ent conditions

before and

after its

passage

through the

membrane. Ionic

conditions

and

the chaperones

that are

present

are

different in

the cytosol

and in the mitochondrial

matrix. It

is

possible

that

a mitochondrial

protein

can

attain its

mature

conformation

only in

the

mitochondrion.

CHAPTER

10

Protein

Locatization

9rz

uelsAs uorlplolsupll

Jo

edfl

laqlouV

rioldtu3 seuosrxoled

6l'0I

pauodrur

aJp

l€ql

suralord

'dlpuouelsuerusod

sJnJJo saruosrxorad o1 suralord;o

uodsue{L

'seurosrxoJJo

eql

ale

-raua8ar

ol salJas

lpql lupuueJ

Jluos

sI ereql

lpqt

^lllrqrssod aqt epnlJxe ol

pJeq

s1

tt

'uaas

z(p

-see

Jq

louupJ

,{aql uaqm uala

'selpoq

Juerq

-ureur,{tdrua-stsoqE

leruosrxorad

qlrm s11at

Jql sJAeJI surxorad

Jo

aluasqp

aq1

'seseJ

Jlrros

lspal lp

uI

'sluauodruoJ

rlaql rnor! 0^0u

ap uJa\f

alquesse o1 alqrssod aq

1q31u

i1

rJqleqM

Jo

uop

-sanb

aql

pJsreJ

tJAJMoq'sl1nsal esaqJ'seruosl

-xorad

Suusxaard

Jo

uouerrdnp

z{q

r(po astre

ueJ saruosrxorad'sellaueBro

papunoq-JuPJq

-ruJru

Jeqlo e>l{

'lpql

paunsse

ueeq.dleraua8

seq

lI

'sJruosrxo,rad;o

aruereaddear

eql

o1 spPJI

aua8 ad,,{.1-plrm

e

Jo

uolpnpoJlul

'sesp)

aseql

Jo

eruos u1

'saua3

asaql

Jo

Jruos uI suollelnru

IInu

a^pq

teql

sllal ruorJ

luesqe

aq ot readde

sJruosrxoJJ4

'saua8

rr;tlads

qllu

palJlluapl

eJB

qJrqm

Jo

tsoru

'sdnor8

uollpluJrualdruol

JAIaMI o1

paddeur

ueaq

aleq saseeslp

leruosl

-xorad

ueunq

pue

'pelllluJpl

uJJq JAeq

sulxo

-rad

ro; Surpor

sauaE aarql-r{1ua,r41

'sulxorad

pJIIeJ

ere uollplrrJoJ aruosrxorad

ro;

parmbar

aJp

lpql

surJtoJd'1oso1b

Jql IIToJJ

p:uodun are

aruosrxorad Jql

Jo

sluauodruor

eql

Jo IIV

'uollJunJ

eruoslxo

-rad

ut sJepJoslp ol

pe>lulT

uJeq aleq

sasPJSIp

upunq

E I<

'sauosxorad;o

>pe1 ,{.q

pasneJ

aq

01

punoJ

se,tlr aruoJpuz(s

ra8a,,vr11a7

Jo

Jseesp

leleJ

eql a)uIS

'llJl

3q1 IoI

IPIJnD

Jre seIlIAIlJe

JIaqJ

'salpJlsqns

reqlo

Jo

z(larren

p

ezIpIXo

ol

eplxo

-rad

uaSorp,{.q eql sJSn uaqt

Jselele)

'sJleJtsqns

rrrorJ

sruolp uaSorpzlq Sur.l,oruar

,{.q aptxo

-rad

uaSorplq ol uaS.dxo

ueluoJ

qJIqM

'uorlez

-q1tn

ua3.,{xo

qll,lr

peuJeJuor

saur,{.zuJ

uIPluoJ

Laql

'auerqruaru

a13urs

e ,{q

pasopua

(rataruep

rud

E'I

01

S'0)

serpoq

ilerus

Jre satuoslxoJad

'losoilr

aql

ol ulnlot ueq]

PUP

uralord

alerlsqns e 6uu{ler auostxotad

aq}

olut

algnqs

leql

sutalold rtlosolfir ale

sloldarar

aql

r

'eruanbas

ZSld

aLll

sputq d1xa6 tolderar

aql

pue

eruanbas

ISI_d

oql

spurq dgxa6 roldara.r

eql

.

'snururel-N

aq1

1e

eruanbas

ZSld

P io snutulal-l

aq1

1e

aruanbes

ISLd

e raqlra

a,req

Aaql

r

'elPls

paploJ

^llnJ

llOql ut sauostxOlAd OlUt

peiloOult

OlP

su!0|016l

r

uals^s

uor.lPlolsuPrl

Jo

adfi1 raqlouV

'rg-zzu\L

01

!\l01

LuolJ ^lllaltp

palaJsuerl

aq

feru uLelord

6uLterolsuelly

g?'fi1

]Hn$Ii

'rS-ZZul

o1 suLalord

selPl

€I-Sllltl

puP

'xalduol

11111

raqla

01

hlOI

LUor] sutalold

salel

0l-6l.utl

I''ot lun$IJ

Aoldtu3 sauostxoJed

-JeJeqJ

esoql

o1 lelrurrs

JJe erJJlJpq

ruorl

uorl

-aJJes

Jo

slusrueq)aur

JqJ

'llJ)

aql uorJ

pslJJJes

eq ol ro

adolJ^uJ

aql

ur Jprser

ol urspldol^J

Jql ruorJ

peuodxJ

Jte

suretoJd

.ruseldlJad

Jql

pJIIer

sr uJql

UJJ^^]aq

eJeds

JqJ'sJJ^el

JupJq

-uIaul

o,e\l

Jo

slslsuo)

JdolJ^uJ

IerrJttpq

JqJ

'sulsru

Pqlau

leuorlPlsuPllol

pue

lpuorlplsuplllsod

qloq

asn saueiquaul

q6norql

ro

ol

pailodxo

ele

leql

sutalold

leua|lpf

o

uoqelolsuejl

leuorlPlsuPrllsod

puP

lPuorlPlsuPllol

LlloB

asn

PuallPB

@

0,r0u ap

alqruJsse

re^e

uet

seruosrxorJd

raqlaq,u

Jo

uortsJnb

eqt uo

sJeeq

srLIJ

'eueJqueu

aqt

ur ^peJJIe

sr

tpqt

d€xJd

qtl.t,r

sDeJJtul

tr

sdeqra4

'aueJqruJtu

eqt sJJlue

1l

111\oq

;o

uortsanb

Jql

sJsrpl qJIqM

'sJdru

uMo

slr seq

d5xa4

'sJupJqruJur

ipurosrxo

-red

ur

punoJ

lou

aJe

surJloJd

Jaqto

J)uasqe

slr ut

Jsnpf,eq

'ura1ord,{a1

e

aq Leu

dgxa4

.uortel8Jlur

Jo

ssaJoJd

eqt

tnoqe

unl.ou>l

sr Jlltll

tnq

,SJdru

eql

pJIIpJ

aruanbas

p

JAerI

JueJqurJru

Ieurosrxo

-rad

aqt

olur

pale;odro)ur

JJe

lpql

sureloJd

'1oso1f:

aql

ol su.rnlet

uotll

pue

,auosLxotad

aq1

olur auplquau

eql

ssoltp

1r

seu.lpl

,1osoy{r

aql

ur urel

-ord

alerlsqns

e spuLq

tolderat

d9xa6 eq1

,

:-

:

!

:i,r.:::

,

.

uotlPzrlel0l

urelold

'rea1r

1a^d tou

aJe sseJoJd

godsuerl

aq1

;o

slrelJp

aLIJ

'uaunl

eq1 otut

uodsuerl

Jo

ssJf,oJd

aql

qlrM

pJAIoAur

are surxorad

JJqto

le]alas

uaqt

pue

'xaldruor

slqt

qtrm

>lJop

sroldalar

aq1

'dg

1xa4

pue

dy1xa4 yo

slsrsuor

qtlqm

'xaldurot

uralord

JuerqluJur

Jrxes

Jql

qlr,tlr

lJpJalur

qloq

d4xa4

pue

dgxa4

JJJqM

'auerquJru

Ieruosrox

-rad

aql

le

JBrJAuo) s.,{e,nqled

godrur

aq;

'snJIJnu

Jql olur

uodrur

JoJ

rualszi.s

rer-rJpJ Jql

salquesJJ Jor^eqJq

3ug11nqs

srql

'ap.dr

raqtoue

e>lpuJpun

o1

1osoilr

aqt ol

suJnlJr

uaqt

pue

TouJlur eql

olur JueJqrueu

aql qSnorqt

tl

rlll.tr sJlotu

'aruosrxorad

ar{t ol

tl

se>lP]

'10so1Lr

Jql ur urJlord

alerlsqns

p

spurq

roldarar

Jql

leql

sMoqs

i

i:

1ij,

lriiti,;ii

'losol^J

eql

pue

aurosrxorad

Jql

ueeMlJq Surlrrb

Le,ra

Jtues aql

ur aneqaq,{aqJ

'sJruosxorad

qlrrr,r pale

-rJosse

uorlrodord

Ileurs

e

.,{.1uo qtrazr

'rrlosolzb

,

1a8re1

JJe raqteJ

1nq

'suralord

auerquaru

ler8

-Jtur

tou

are

sroldarar d4xa4

pue

dgxa4

aq1

'JuprqruJru

eql

qlr^t

sJlerJosse

1l

uJqM ulaloJd

JleJlsqns

eql

punoJp

sJIquJSSe

IJuueqJ

Jql

rpql

asoddns

ol

pue

pepr

plo

ue

lf,eJJnseJ

ol sr dlr

-gqrssod

JUO

'srqt

tlrurad

ol

puedxa

plno)

Ieu

-uBqr

Surlsrxaard

e

Jo

Jrnlluls Jql 1!|or{

Jeell

lou

sr

u

'sprJp

ourure

Jo

peeJql pJploJun

ue

01

uqe

Surqlaruos

ur allaue8Jo

aql

olur eueJqureur

Jql ur

IJuueqJ

e

q8norql

sassed

U

ereqm

'uorJpuoq)otrru

Jo

UE

Jql otur

a8essed JoJ

urJl

-ord

e

p1o;un

ot

tuauarrnbal

aql

qtl.tt

stseJluoJ

sIqJ

'elets

peplol

u(11n;

'arnteur

Jleql uI

euos

-rxorad

Jql olur

palrodur

aq

upf, surJloJd

'sa11aue8ro

Jaqto

otur

uodsuert

roJ

pesn

rualsLs

aqt

uroJJ

sJJuJJa;;p

tueuodur

>lJpru

leql

sJlnteeJ

Iensnun

spq Jtuosrxorad

aql

olul

uodsue41

'uorlJPJr

uorleJolsueJl

Jql qlr,t,r pauJaruoJ

saxalduror pJlerJosse

-eueJquaru

Jo

tred

are suralord

raqlo aq1 ,r(1anr1

-radsar

'd4xa4

pue

dgxa4

pJIIeJ

JJe sleu8rs;o

sad,{t

o.,r,l1 Jql

purq

teqt

sJoldJlar

leuosrxorad

aq1

'1osol,b

eqt

ruoJJ surato,rd

Jo

uodur

Jql JoJ

,dressalau

are

suralord

leuosrxorad

IprelaS

'€SJd

prllpl

aluanbas

Jo

ad.{l

prrqt

e rq

,{eur

araql

alqrssod

sr

tI

'^lleurJlur

Jo snurruJeJ

-N

Jqt Jeeu pJlef,ol

Jq

upJ srql

pue

'AtrsrJ^lp

qlnu

qlru

ure8e

'spoe

ourrue Juru

Jo

aruanbas

e

sr

leu8rs

ZSJd

JqJ'alaue8ro

eqt olur

uodur

Jleql

JJnsuJ

ol

luerJrJJns

sr suralord

rrlosofr

Jo

snurluJJl-)

aqt ol atuanbas

Jlqplrns

e

Jo

uorl

-1ppp

eql

'leu8rs

ISId

e se

tre

ot uMoqs

uJJq

JAeq

sJJuJnbas

Jo

^,{larren

a8rel

e ,lrou

lnq'(na1

-s,{1-rag)

1;5

aruanbJs

aql se

pezrJJtJpJpqJ

Lleur8rro

se,lr

tI

'snurulat-l

Jql

le

aprtdadettal

ro

-rn

e sr

leu8rs

ISJd

aqJ

'ZSJd

pue

I(SJd)

leu8rs

SurtaSret

lerurxorad

pJIIeJ'saruanbas

UOqS

OMl

JO

JJqIIJ

JAEq

XIJ]EIII

Jql OIUI

0r

utldvHl

9rz

terized

for

eukaryotic cells, and we can recog-

nize

some

related components.

litriiiii: !t.-i.,1.

shows that

proteins

that are exported from the

cytoplasm

have one of four fates:

.

to

be inserted into the inner membrane,

.

to be translocated through the

inner

membrane to rest in the

periplasm,

.

to be inserted into the outer membrane, or

o

to be translocated through the outer

membrane into the medium.

Different

protein

complexes

in

the

inner

membrane are responsible for transport of

pro-

teins depending

on whether their fate is to

pass

through

or stay within the inner membrane.

This resembles the situation

in mitochondria,

where

different complexes

in

each of the

inner

and outer

membranes handle different subsets

of

protein

substrates

depending on their desti-

nations

(see

Section 10.16,

Posttranslational

Membrane

Insertion Depends on Leader

Sequences)

A

difference

from import into

organelles

is that transfer in E. coli may be either

co- or

posttranslational.

Some

proteins

are

secreted

both cotranslationally and

posttrans-

lationally,

and the relative kinetics of translation

versus

secretion through the

membrane could

determine

the balance.

Exported bacterial

proteins

have

N-terminal

leader sequences

with a hydrophilic

N-terminus

and

an adjacent

hydrophobic

core.

The Ieader

is cleaved by

a

signal

peptidase

that recognizes

precursor

forms of several exported

proteins.

The signal

peptidase

is an integral membrane

protein

located

in

the

inner membrane. Muta-

tions

in

N-terminal

leaders

prevent

secretion;

they are suppressed by

mutations in other

genes,

which

are thus defined as components

of

the

protein

export

apparatus. Several

genes

given

the

general

description sec are

impli-

cated

in coding for components of the secre-

tory apparatus

by the occurrence of mutations

that

block secretion of

many or all exported

proterns.

@

The Sec System

Transports Proteins

into

and

Through

.

The bacteriat SecYEG

transtocon in the

inner

membrane is related to the eukarvotic

Sec61

transtocon.

o

Various chaperones

are involved

in

directing

secreted

proteins

to the translocon.

i

1r,i.liir.1

r

rr

r,',

BacteridI

proteins

may be

exported

ejther

posttrans-

Lational"Ly or cotranslationat[y.

and

may be [ocated

within

either

membrane or the

periplasmic

space.

or

may be secreted.

i:li;iiiiii r,

:1

,i,':

The Sec system

has the

SecYEG

trans[o-

con embedded

in the

membrane.

the

SecA-associated

pro-

tein that

pushes proteins through

the channet,

the SecB

chaperone

that transfers

nascent

proteins

to SecA,

and the

signal.

peptidase

that cleaves

the

N-terminaI

signaI

from

the transtocated

protein.

There are

several

systems

for transport

through

the

inner membrane.

The

best characterized

is

the Sec system,

whose

components

are shown

in

i ii,i.ii;i

i:,

,,',.

The

translocon

that

is embed-

ded

in

the

membrane

consists

of

three

subunits

that

are related

to

the

components

of

mam-

malian/yeast

Sec6

I. Each

of the

subunits

is an

integral transmembrane

protein.

(SecY has ten

transmembrane

segments;

SecE

has three')

The

functional

translocon

is a trimer

with one

copy

of each

subunit.

The

major

pathway for direct-

ing

proteins to the

translocon

consists

of SecB

and SecA.

SecB

is a

chaperone

that

binds

to the

the Inner

Membrane

10.21 The sec system

Transports

Proteins

into and

Through

the

Inner

Membrane

247

;li.:i..:Tl

I

""-::

SecB/SecA

transfer

proteins

to the

transto-

con

that

pass

through

the

membrane.

4.4S RNA

transfers

proteins

that

enter the membrane.

nascent

protein

to control

its folding.

It trans-

fers

the

protein

to

SecA, which

in turn

transfers

it

to the

translocon.

r:j..ii:lii:

ri-.,irir,

Shows

that there

are two

pre_

dominant

ways of

directing

proteins

to

the Sec

channel:

.

the SecB

chaperone,

and

.

the

4.5S RNA-based

SRP.

Several

chaperones

can increase

the

effi-

ciency

of

bacterial

protein

export

by

preventing

premature

folding;

they include

"trigger

factor"

(characterized

as

a chaperone

that assists

export),

GroEL

(see

Section I0.5,

Chaperones

Are

Needed by

Newly

Synthesized

and

by Dena-

tured

Proteins,

and Section

10.I8,

Inner and

Outer Mitochondrial

Membranes

Have

Differ-

ent Translocons),

and

SecB

(identified

as the

product

of

one of

the sec mutants).

SecB is the

least

abundant

of these

proteins;

however,

it

has

the major

role in

promoting

export. This

role

comprises

two

functions:

first,

SecB behaves

as

a chaperone

and binds

to a nascent protein

to retard

folding.

It

cannot

reverse

the change

in

structure

of a folded

protein,

so it

does not

function

as

an unfolding

factor.

Its role

is there-

fore

ro inhibit

improper

folding

of the

newly

synthesized protein.

Second,

SecB has

an affin-

ity

for

the

protein

SecA.

This

allows it

to target

a

precursor

protein

to

the membrane.

The SecB-

SecYEG pathway

is

used

for translocation

of

proteins

that

are secreted

into

the

periplasm

and is

summarized

in

ii{li:i:i:

.:

r:.4?.

SecA is

a large

peripheral

membrane pro-

tein that

has

alternative

ways

to associate

with

the

membrane.

As a

peripheral

membrane

pro-

tein,

it associates

with

the membrane

bv

virtue

Protein

Localization

FIS{JRf

1*"47

SecB transfers

a nascent

protein

to SecA,

whjch inserts

the

protein

into

the channet.

Translocation

requires

hydrotysis

ofATP and

a

protonmotive

force.

SecA

undergoes cyctes

of association

and dissociation

with the

channel and

provides

the motive

force

to

push

the

pro-

tein through.

of its affinity

for acidic

lipids

and for

the Secy

component

of

the translocon,

which

are

part

of a multisubunit

complex

that

provides

the

translocase

function.

In

the

presence

of other

proteins

(SecD

and

SecF), however,

SecA can

be found

as a membrane-spanning

protein.

It

probably

provides

the motor

that

pushes

the

substrate

protein

through

the SecYEG

translocon.

SecA recognizes

both

SecB

and the

precur-

sor

protein

that it chaperones;

most likely,

fea-

tures

of the

mature

protein

sequence

as well

as

its leader

are required

for recognition.

SecA

has

an ATPase

activity

that

depends

upon

binding

to

lipids,

Secl and

a

precursor

protein.

The

MPase

functions

in a

cyclical

manner

during

translocation.

After

SecA binds

a

precursor

pro-

tein

it binds

ATP,

and

-20

amino

acids

are

translocated

through

the membrane.

Hydroly-

sis of ATP

is required

to release

the

precursor

248

CHAPTER

10

from

SecA.

The cycle may

then be repeated.

Precursor

protein

is bound

again to

provide

the

spur to bind

more ATP,

translocate another seg-

ment

of

protein,

and release

the

precursor.

SecA

may alternate between the

peripheral

and inte-

gral

membrane forms during translocation;

with

each cycle. a

30 kD

domain of SecA may insert

into the membrane and then retract.

Another

process

can also undertake translo-

cation.

When

a

precursor

is released by SecA,

it can be driven

through

the membrane by a

protonmotive force

(that

is,

an electrical

poten-

tial across

the membrane). This

process

cannot

initiate transfer through the membrane, but

it

can continue

the

process

initiated by a cycle of

SecA ATPase action.

Thus

after or between cycles

of the SecA-AIP

driven reaction, the

protonmo-

tive

force

can

drive

translocation

of the

precursor.

The E. coli ribonucleoprotein complex of

4.5S RNA with

Ffh

and

FtsY

proteins

is

a coun-

terpart to

the eukaryotic SRP

(see

Section 10.9,

The SRP

Interacts with the

SRP

Receptor). It

probably plays

the

role

of

keeping

the

nascent

protein

in an appropriate conformation until

it

interacts with other components of

the

secre-

tory apparatus.

It is needed for the secretion of

some,

but not all,

proteins.

As we see

in

Figure I0.46, its substrates are integral

mem-

brane

proteins.

The basis for differential selec-

tion

of substrates

is

that the E. coli SRP

recognizes

an anchor sequence

in the

protein

(anchor

sequences

by definition are

present

only

in inte-

gral

membrane

proteins).

Chloroplasts

have

counterparts

to

the

Ffh and FtsY

proteins,

but

do not require an

RNA

component.

fj-lri.:lii: I

i,r

iir

Ml,3 coat

protein inserts

into the

jnner

membrane by

making

an initiaI

etectrostatic

contact.

fot-

I'owed by

jnsertion

of

hydrophobic

sequences.

Transtoca-

tion

is

driven

by

hydrophobic

interactions

and

a

protonmotive

force

untiI the

anchor

sequence

enters

the

memDrane.

cation

apparatus!

It

can insert

posttranslation-

ally into

protein-free

liposomes.

Targeting

the

protein

to the

membrane

requires

specific

sequences

(comprising basic

residues)

in

the N-

and C-terminal

regions

of the

protein. They

may interact

with

negatively

charged

heads

of

phospholipids. The

protein

then

enters

the

mem-

brane by

using

hydrophobic

groups in its

N-terminal

leader

sequence

and

an

internal

anchor sequence.

Hydrophobicity

is the

main

driving

force

for translocation,

but

it can

be

assisted by

a

protonmotive

force that

is

gener-

ated between

the

positively charged

periplasmic

side of the

membrane

and an

acidic

region

in the

protein.

This drives

the

protein through

the

mem-

brane, and

leader

peptidase

can then

cleave

the

N-terminal

sequence.

The

generality of

this

mech-

anism

in bacteria

is unclear;

it

may apply

only

@

Sec-Independent

Translocation

Systems

in E. coli

o

E. coli and organetles

have related

systems

for

orotei n tra

ns[ocation.

o

One system

al[ows certain

proteins

to insert

into

membranes without a transtocation apparatus.

.

YidC is homologous to a

mitochondrial

system

for

transferring

proteins

into

the

inner membrane.

r

The tat system transfers

proteins

with a twin

arginine

motif

into

the

periplasmic

space.

The most striking alternative system

for

pro-

tein translocation

in E. coli is revealed by the

coat

protein

of

phage

Ml3.

iil"':"ifiil

liil"+i; shows

that this does

not appear to require any translo-

10.22

Sec-Independent

Transtocation

Systems

in E. coli

249

to the special

case of

bacteriophage

coat

proteins.

Some

chloroplast

proteins

may insert

into the

thylakoid

membrane

by a

similar

pathway.

Mutations

in the

gene yidCblock

insertion

of

proteins

into the inner

membrane.

YidC is

homologous

to the

protein

Oxalp

that is

required

when proteins

are inserted

into the

innermitochondrial

membrane

from

the matrix.

It can function

either independently

of SecYEG

or in

conjunction with

it. The

insertion

of some

of the YidC-dependent proteins

requires

SecYEG,

which

suggests

that YidC

acts in

conjunction

with the

translocon

to divert

the substrate into

membrane

insertion

as opposed

to

secretion.

Other

proteins

whose insertion

depends

on YidC

do not require

SecYEG:

It seems

likely that

some

other

(unidentified)

functions

are required

instead

of the translocon.

The

tat system

is named

for its

ability to

transport

proteins

bearing

a twin arginine

tar-

geting

motif. It

is responsible

for

translocation

of

proteins

that have

tightly

bound

cofactors.

This

may mean

that

they have limitations

on

their

ability to

unfold for

passage

through the

membrane.

This would

be

contrary to

the

principle

of most translocation

systems.

where

the

protein

passes

through

the membrane

in

an unfolded

state and

then must

be folded into

its

mature

conformation

after

passage.

This

sys-

tem is related

to a system in

the chloroplast

thy-

lakoid

lumen

called Hcf 106.

Both

of these

systems

transport

proteins

into the

periplasm.

@

Summary

A

protein

that is inserted

into,

or

passes

through,

a membrane

has

a signal

sequence

that is recog-

nized

by a receptor

that is

part

of the membrane

or that

can

associate

with it. The

protein

passes

through

an aqueous

channel

that is

created

by

transmembrane

protein(s)

that

reside in

the

membrane.

In

almost

all cases.

the

protein

passes

through

the

channel in

an unfolded

form,

and

association

with chaperones

when it

emerges is

necessary

in

order

to acquire

the

correct confor-

mation.

The major

exception

is

the

peroxisome.

where

an imported protein

in

its mature

confor-

mation

binds

to

a cytosolic

protein

that carries

it

through

the

channel

in the

membrane.

Synthesis

of

proteins

in

the cytosol

stafis

on

"free"

ribosomes.

Proteins

that

are secreted

from

the

cell

or that

are inserted

into

mem-

branes

of the

reticuloendothelial

system

start

with an

N-terminal

signal

sequence

that causes

the ribosome

to become

attached

to the mem-

CHAPTER

10

Protein

Localization

brane of the

endoplasmic reticulum.

The

pro-

tein is translocated

through

the membrane

by

cotranslational

transfer. The

process

starts when

the

signal sequence is recognized

by the SRP

(a

ribonucleoprotein particle).

which interrupts

translation.

The

SRP binds to the

SRP receptor

in

the ER membrane

and transfers

the

signal

sequence to

the Sec6l/TRAM

receptor

in the

membrane.

Synthesis resumes,

and

the

protein

is

translocated through

the membrane

while it

is being

synthesized, although

there

is no

ener-

getic

connection

between

the

processes.

The

channel

through the membrane provides

a

hydrophilic

environment

and is largely

made

of the

protein

Sec6l.

A

secreted

protein passes

completely

through the membrane

into

the ER lumen.

Pro-

teins

that are integrated

into membranes

can

be

divided into two

general

tlpes

based

on their

orientation. For

type I integral

membrane pro-

teins, the

N-terminal signal

sequence

is cleaved,

and

transfer through

the membrane

is

halted

later

by an anchor

sequence.

The

protein

becomes

oriented in the

membrane

with its

N-terminus on the far

side and

its C-terminus

in

the cytosol.

\pe

II

proteins

do not

have a

cleavable

N-terminal signal,

but instead

have

a

combined

signal-anchor

sequence,

which

enters

the membrane

and becomes

embedded

in it.

This causes

the C-terminus

to be located

on the

far side, whereas

the

N-terminus remains

in

the

cytosol. The

orientation

of the

signal-anchor

is

determined

by

the

"positive

inside"

rule,

which

states that

the side

of the anchor

with more

pos-

itive

charges

will be located

in the

cytoplasm.

Proteins

that have

single transmembrane

span-

ning regions

move laterally

from

the

channel

into the lipid

bilayer.

Proteins

may have

mul-

tiple membrane-spanning

regions,

with loops

between

them

protruding

on

either

side of

the

membrane.

The

mechanism

of insertion

of mul-

tiple segments

is unknown.

In the

absence

of any

particular

signal,

a

protein

is released

into

the cytosol

when

its syn-

thesis is

completed. Proteins

are imported post-

translationally

into mitochondria

or chloroplasts.

They

possess

N-terminal leader

sequences

that

target

them

to the

outer membrane

of tne

organelle

envelope;

they then

are transported

through

the outer

and inner

membranes

into

the matrix.

Translocation

requires

ATP

and a

potential

across the

inner

membrane.

The

N-terminal leader

is cleaved

by

a

protease

within

the

organelle.

Proteins

that reside

within the

membranes

or intermembrane

space

possess

a

signal

(which

becomes

N-terminal

when

the

250

Lewin Benjamin (ed.) Genes IX

Подождите немного. Документ загружается.