N a t i . S t r i c t u r e a n d f u n c t i o n o f S i g n a l R e c o g n i t i o n P ... inhibit*
- t h e f a i i b e i e l o n p a b o n nj {he n ; ^ u i i i ... ^RP ' '4 a- d :fi u i v d '
',•,;„ >t is ; t ' :4 hi. '1992) •J
BA.
Nati.
Stricture and function of Signal Recognition Particle ( S E P )
llvntu h I PU.ke A- Brnsbard f »obber-1eni /cut run i jut Miilc'h uiatf lUolnvsc fh'idriht rv. Itn Siurtih< in;- t hut S
A c i \\
si,.,s".^
r, ,iU)- . t i : n . ! k . i l e d
«».i).o I-f!
S R P M JI>C
/ i.'.'' b.oie.'-i- c'.;c
p4>, ;ht
e i t h e r a>
S R P can
144 rv.* \ j . v
• * • ' . i'.i >
\ ' .
d'.e s t e m and l o o p oi t h e "S R N \ t o which mctlicic S R P I'* bunls f i : , 'A] gests tl
d";> MI- I , m i - o s ' P , u a s . •! ;} e r - T i . ! : ! , ; " , " 'tc:;
cparafrd b r; ic-anxtn. >* P " | \ p c p n d c
aithcieni I'm buulint; S R P M to the S R P R N \ nascen ^UP-
;P:;S .mlc- r, >p-^ena*o;nni* condmon » W hen die S
R P p:otein> ;rv expressed/>< o.'/o from i^cu
. L)N \•> t, ^KRft.s h u u K to ? S R N A in die
than intact S R P 5 4 or S R P [37, Bacher and intact D o b b e r s t e t n , unpublished observations). A sig- The nal sequence binding p o c k e t has been proposed inhibit
absence T SR P "2•> ;a i highly positr, ei\ c h u n v d
to be formed by the hydrophobic faces of the the RT
region, rear :hc N-k*iminus S R P 7 ? *r11 >
amphipathic helices predicted to be present in [20] ai
assemble- into a ...omplex vuth 7S R N \ in the
•sRP""4\I. d h e p o c k e t would b e lined by many domai
n r c - e n e e o f S R P P H '1 he tv.o /« .urn \\nthesi/cd
methionine residues w h i c h , by virtue of their The in
proteins d o n*a. oi d o \er> inefficienti's, associate
hneat h y d r o p h o b i c side chains, could readily plex o
usth each was f o u n d with the mammalian
G T P a s e d o m a i n (similar to small G T P a s e s ) with site [
components in 1 nro \ 21 ] arid with die homolo-
an N-terminal e x t e n s i o n [ i, 26]. S R P 5 4 N + G "nine
gous yeast components /// \i\ firs S a returnie\
may be c l o s e to S R P 5 4 M since the alkylation of { D P )
[ s j j . N o reuuifement lor S R P I V has been found
S R P 5 4 N + G with N-ethy! maletmide ( N E M ) D P
in the in vara assembly of m a m m a l i a n S R P 5 4
prevents the b i n d i n g o f a signal sequence to dispo
with the ^mailer 7S R N A homologues oi E cull
S R P 5 4 M [16]. T h i s , a n d the finding that the unit
(4,5 S R N A ) and H .uhlilis (US R N A ) which lack
binding of a signal sequence to S R P 5 4 M is Muta
145
ich
inefficient in the absence o f S R P 5 4 N + G , sug-
b o u n d by D P a u p o n interacting with the c o m p l e x
gests that S R P 5 4 N + G m a y control the affinity
o f r i b o s o m e , nascent p o l y p e p t i d e and S R P and
by which S R P 5 4 M b i n d s a signal sequence. N o
that this m a y trigger the release o f the signal f r o m S R P as well as an inc reased
requirement for G T P has been detected for the
i of
binding of S R P to ribosome/nascent chain c o m -
affinity between S R P a n d D P [23]. U p o n h y d r o l -
plexes. H o w e v e r , the release of S R P f r o m such
ysis o f G T P , the interaction between D P a n d
c o m p l e x e s requires G T P [ 2 ] .
S R P b e c o m e s sensitive t o salt extraction [3].
;wo
U s i n g reconstituted S R P , c o m p o n e n t s h a v e
:hl>
been identified w h i c h are necessary for mediating
on-
The SRP-mediated pausing of elongation
the targeting o f a nascent secretory protein to m i c r o s o m a l m e m b r a n e s [30]. Selective a l k y l a -
or
tion o f S R P 6 8 / 7 2 in S R P a l l o w e d the modified
and
O n c e S R P has b o u n d to a signal sequence in a
NA
nascent polypeptide, it c a n retard or even stop
S R P to still bind to a c o m p l e x o f r i b o s o m e a n d
>ro-
the further elongation [ 3 4 ] . T h i s activity o f S R P
nascent polypeptide a n d to arrest the further
3hi-
involves the A l u d o m a i n o f S R P [28]. R e c o n s t i -
elongation but prevented the interaction o f the
to
tution o f S R P which o m i t s S R P 9 / 1 4 , or includes
c o m p l e x with the m e m b r a n e [ 3 0 ] . Furthermore,
l
alkylated SRP9/14, renders the resulting particles
the S R P 6 8 / 7 2 - a l k y l a t e d S R P displayed a re-
has
unable to inhibit the elongation [30]. N e v e r t h e -
d u c e d affinity for D P . S R P l a c k i n g S R P 5 4 N + G
10],
less, such particles, and even S R P lacking the
likewise failed to p r o m o t e targeting and to f o r m a
:cif-
entire A l u d o m a i n , are still capable of p r o m o t i n g
stable c o m p l e x with D P [36]. I n d e e d , S R P 5 4
1 on
the co-translational translocation of a secretory
appears to interact directly w i t h D P , as a G T -
nity
protein in vitro, albeit with lower efficiency than
Pase activity w a s greatly e n h a n c e d w h e n b o t h
and
intact S R P [29].
proteins were incubated in a purified system in
sig-
T h e m e c h a n i s m by w h i c h the elongation is
the presence o f S R P R N A [ 2 2 ] . T h u s it appears
)sed
inhibited is u n k n o w n . It h a s been p r o p o s e d that
that molecules required to m e d i a t e a stable inter-
the R N A in the A l u d o m a i n resembles a t R N A
action o f S R P with the m e m b r a n e , involve o n the
[20] and m a y thus exert its inhibitory effect in the
o n e h a n d S R P 5 4 and S R P 6 8 / 7 2 , a n d o n the
the t in any
d o m a i n o f the r i b o s o m e t o which t R N A s bind.
other D P and possibly other m e m b r a n e c o m p o -
heir
T h e inhibition o f elongation lasts until t h e c o m -
nents.
idilv
plex o f S R P , ribosome a n d nascent p o l y p e p t i d e
i
interacts with the D P in t h e R E R m e m b r a n e [4]. The release of the signai sequence from S R P
goal thin L the
Docking of S R P to the R E R membrane
:y be
M o s t recently, the reconstitution and functional characterization o f m e m b r a n e vesicles f r o m d e -
RP.
W h e n S R P in the c o m p l e x with r i b o s o m e and
tergent solubilized and fractionated m i c r o s o m e s
like
nascent chain interacts w i t h the m e m b r a n e of the
h a s led to the identification o f essential c o m p o -
:P54
R E R in the presence o f G T P , the signal sequence
nents o f the translocation m a c h i n e r y in the R E R
is released from S R P 5 4 a n d m a k e s c o n t a c t with
m e m b r a n e [5, 6]. O n e such c o m p o n e n t essential
cted
Sec61p, a protein c o m p o n e n t o f the translocation
for the translocation o f m o d e l polypeptides w a s
with
site [ 1 1 , 6 ] . T h e c o m p o n e n t in the R E R m e m -
identified as the m a m m a l i a n S e c 6 1 p [6]. T h i s
-f G
brane to which S R P b i n d s is the d o c k i n g protein
protein h a d previously been identified as the
( D P ) or S R P receptor.
m a j o r R E R m e m b r a n e protein in contact w i t h
>n o f EM)
D P is c o m p o s e d of a largely cytoplasmically
different signal sequences, o n c e they h a d been
e to
disposed a - and a membrane-integrated /?-sub-
released f r o m S R P [ 1 1 ] . W h e n m e m b r a n e vesi-
. the
unit [13, 32] both of w h i c h bind G T P [2].
cles lacking sec61p were added to a translocation
4 is
M u t a t i o n a l analysis o f D P a suggests that G T P is
assay, the signal sequence r e m a i n e d b o u n d to
146 i ' • - O n , -».. , ,( 0 • w f i ' > d ' J ) • u
a.,
* " > i u , - a a ». r-f.'i,, I A ; ,i''i,>-:o K . ' * " . ' ' ) ( oil ^; >*>o ( , , / ) 1
S U P in oilier o r g a n i s m s
4 i n s ' , ' : ! , i , R . t p u - i s . • i ' ! A ( n i m o u - R i | 'iu 11 , c n , r ' ' ' 11' i !; ;
i < I !!>'.)! Hi* li A B i . i H -I I , j I '>\ M OH M ' I t ' T N l i . . .,>,.,, , , | | , ! ; . S K I ' R \ \ h . H C U . V f l d i >(„ r ; P e d i.' c 1 .'*- > ! a »r. h pi i f f
mil v
;> I », It u i - ! , , n ; . i , P ' - h n S A. R ^ p o p o s i t \
ieo o,o>-: u , , e , s a \
i ! o t ,S , - napoUf.« M I ,
i l ' « ' 0 ,t ^, in,;,- ' - i ' < . n u n >i n l ' „ ' l « r N t( iO-n ICM i , k i l i c , k i A R i f v
%„V
•v,! i ! \ ( I ' " ) ' n H , i| ' ! 0,')
, ' "*"' I, a a i o . i t o , - a h o r a o i < > e o 11 -> t o S K P It \ \ , • fX'Cll
4l SI n o F5'", s i . j h i u ' ( 1 A VV^io-f p | ! ' ) • ) ' , N a t u r e ! K> sr:'..sa
f h c S R P o f / c o m i i r h t h e i< kDd
k o n i o t o v e .>f*SRP5i sp-W >M Fih) which is
round
; o H u z n S X i > n b h e f , r n n }{ , t ' > 0 | ) f ( c i l B i o l 1 1 >
ir, a c n ' n p i c x M i l l tin- 4 5 S K N \ ? . \ . f,
f 12 1 \'
o i l i e r p r o t e i n ha*-
p a f i k l c ,i;
ufdason
to
p
is
been
" u . •> ]
detected
P IK i n t e r a c t
in
fhc
1! H n h S ( i n t l i c h I ) , W . c a n i a n n M . R j p i v p n i t I * \ &
v\ith d i e
! ) o h b e ! s h ' : n B ! i W1 i f ( V!l Bu>! ! P 4 4 I * I ,tr*en N k / , u c b ( n i ) t ) l ) N u d o c \ c i d s R e s 19
•4c n a ! v c q u e r v e o t r. a s * , e r a p r e n r o l a c t i n //? m r o
( 1"'} liiu.
: n / ' < >;«,, rnc proteins j -d |. I h c S R P i! unl
\c".art!
p i o t e i n s , ne
of
which
I a.t'tct t . ( i . u v , < i ' D . If uktii'. R N . ( o u s ' . c t o t , P S i n c h \ W a l t e r I M l ' i K i ) N a t u r e 5 I H !3H
o f ,S , ,>r,>;!>•>lie c o n s i s t « i f o i k R N A m o l e c u l e
< -cR
'! It m n H( X W d u n !' * '/'''U < oii I ' ! 1 4 1
i t t u^r.-ih k *h k / . - v i c h l W c b h I R . M , u s h a l K ; s \ (",
is t i n *
t t . ' h c n P I , U aO-.-r P & D n b b e t a c m B < P ) H X ) N u d c i c
S R P 5 4 l i O i n o l o j M c l f ) j I s k c i n / , ' toh, d i s r u p t i o n i ^ ! uifiiik I, i i h ' j i S , WOIMS I I , < ,mcr \ . t o i l e t v c % i ) &
of ihc S R P in S t t>n>\issue differeutfalh .iflceb. t h e •secri.'Uor. o i
different fM'ofein^. 1
he disruption
o f die iiene j n e o d i n t * S K P ^ 1 ^evefeiy inhibits but
doeN ns> >wrli (*)]. 1
un{>on.int for
hus,
it
protein ^eere-
s s i h f , all o i ^ a r i i M H s .
D o b h c i O c m B ( I ' t ' / ' i \ a m c ^ 9 '41 • 743 U> P ' l t e k c f t , f l s - h S }> 'Miusvh K , \ - h f o u i A.J & D o b h c r .lent H s S W ? i f A I B O J i I 1 > H ~ 1 ^ 1 ! 7 f dhk-i i f P t r l s n s , \ 4 i f o r 4 \ J , R e m u s \ f . f r i n k R & D D h b c r a c i r , H I l ' ) 9 * ) I ( eli f i i o l i ? l T / 7 9 S S 14 V l e v c i 1)1 & P ) , ) b b - ; r , t c ! n B i 19X0) J ( d ! Biol S 7 •MO
sOH
19. X u m t a r i J & W a l t e i I ' i | W ? | O n r O p C e l l B i o l , 4 ; s / i - '.SO ^ H l \a siunal sequcHct'-*»pt*cilic e h a p e r o n e
2 0 . f ) k u i a N (1000j I M o l I v o l ) t sOO s i t ) 2 1 . P h i l l i p s O J Ac S i l h u v y f J C i 99?.) N a t u r e ^59 7 4 4 - 7 4 6
S R P ui sis nimifaal lurni muy be represented by the A. / o h ^RP. B y binding tu the h y d r o p h o b i c core of a ->i'.',na! sequence it maintains it m a
2 2 . P u n t / M A , B e t m . i c i r i H I ) , S t r u b K . "i'opt 1), W i l h e l r n H & W a l t e r P I ! W O I S c i e n c e 2 5 0 1 i 1 1 - 1 117 2 3 . K a p t e j k o P J P,S, Sec \ i interact '.vsih the mature portion of secretory ('prejprolem > and thereby may cooperate with
I c t v e y 1) t l O O O ) ( eli " 0 P 6 0 0 26. R u m i s c h K , W e b b J. f f e t / J, P i c h n S , P i , i n k R , V t n g r o n M i e i n B (1 OHO) N a t u r e UO, 4 7 8 - 4 8 2 2 7 . R O r n i ' c h K W e b b J, f a i i i ' d b a e h K , G a u s c p o h l H & f ) o l ) f M ; r s t o » B f 1 9 9 0 ) J C o l l B i o l . I l l : 1793 - 1 K 0 2
147 28. Siegei V & W a l t e r P ( 1 9 8 5 ) J . C e l l B i o l . 100: 1 9 1 3 1921 29. Siegei V & W a l t e r P ( 1 9 8 6 ) N a t u r e 320: 8 1 - 8 4 30. Siegei V & W a l t e r P ( 1 9 8 8 ) C e l l 52: 3 9 - 4 9 3 1 . S t r u b K & W a l t e r P ( 1 9 9 0 ) M o l . C e l l . B i o l . 10: 7 7 7 784 32. T a j i m a S, Lauffer L. R a t h V L & W a l t e r P ( 1 9 8 6 ) J . Cell Biol. 103: 1 1 6 7 - 1 1 7 8
33. W a l t e r P & B l o b e l G ( 1 9 8 1 ) J . C e l l Biol. 91: 5 5 7 - 5 6 1 3 4 . W a l t e r P & B l o b e l G ( 1 9 8 3 ) C e l l 34: 5 2 5 - 5 3 3 35. W a l t e r P , G i l m o r e R & B l o b e l G ( 1 9 8 4 ) C e l l 38: 5 - 8 36. Z o p f D , Bernstein H D . J o h n s o n A E & W a l t e r P ( 1 9 9 0 ) E M B O J . 9: 4 5 1 1 - 4 5 1 7 37. Z o p f D , Bernstein H D & W a l t e r P ( 1 9 9 3 ) J . Cell Biol. 120: 1 1 1 3 - 1 1 2 1 3 8 . Z w i e b C ( 1 9 9 1 ) N u c l e i c A c i d s R e s . 19: 2 9 5 5 - 2 9 6 0
