SycE allows secretion of YopE-DHFR hybrids by the Yersinia enterocolitica type III Ysc system

Molecular Microbiology (2002)

46

(4), 1183–1197

© 2002 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Science, 200246Original Article

Role of SycE in type III secretionM. F. Feldman, S. Müller, E. Wüest and G. R. Cornelis

Accepted 2 September, 2002. *For correspondence at the firstaddress. E-mail [email protected]; Tel. (

+

41) 61 267 2110; Fax(

+

41) 61 267 2118.

†

Present address: Institut für Mikrobiologie, FMLWeihenstephan, Technische Universität München, WeihenstephanerBerg 3, 85350 Freising, Germany.

SycE allows secretion of YopE–DHFR hybrids by the

Yersinia enterocolitica

type III Ysc system

Mario F. Feldman,

1,2

Simone Müller,

2†

Esther Wüest

1

and Guy R. Cornelis

1,2

*

1

Division of Molecular Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 30–50, CH-4056 Basel, Switzerland.

2

Microbial Pathogenesis Unit, Christian de Duve Institute of Cellular Pathology, Université catholique de Louvain, B-1200 Brussels, Belgium.

Summary

The Ysc type III secretion system allows

Yersiniaenterocolitica

to translocate virulence proteins, calledYop effectors, into the cytosol of eukaryotic cells.Some of the Yop effectors possess an individual chap-erone called a Syc protein. The first 15 amino acids ofthe YopE effector constitute a secretion signal thatis sufficient to promote secretion of several reporterproteins. Residues 15–50 of YopE comprise the mini-mal binding domain for the SycE chaperone. In thisstudy, we investigated the secretion by the Ysc sys-tem of several YopE–DHFR hybrid proteins with differ-ent folding properties, and evaluated the role of SycE,the cognate chaperone of YopE, in this context. Wehave analysed the secretion of hybrids containing 16(YopE

16

), 52 (YopE

52

) and 80 (the complete region cov-ered by the chaperone, YopE

80

) amino acids of YopEor full-length YopE (YopE

FL

) with wild-type DHFRand two mutants with altered folding properties.The hybrids containing DHFR

DDDD

77, the mutant whosefolding properties are the most drastically affected,could be secreted in all the conditions tested, evenin the absence of the chaperone SycE. In contrast,DHFRwt could only be secreted fused to the first 52amino acids of YopE, and its secretion was strictlydependent on SycE. The hybrids YopE

80

–DHFRwtand YopE

FL

–DHFRwt were not secreted. YopE

FL

–DHFRwt completely jammed the channel in an SycE-

dependent fashion. Our experiments indicate that, inorder to be secreted, proteins must be unfolded oronly partially folded, and that TSS chaperones couldkeep their substrates in a secretion-competent con-formation, probably by preventing their folding. Inaddition, they show that the secretion apparatus canreject folded proteins if they are not deeply engagedinto the injectisome.

Introduction

Type III secretion (TTS) is a mechanism used by severalGram-negative pathogenic bacteria to secrete and trans-locate virulence factors or ‘effector’ proteins into eukary-otic cells in order to overcome the defences of the host(Cornelis and Van Gijsegem, 2000). In one of the best-studied systems so far, the

Yersinia

Ysc system, the effec-tors are called Yops. Normally, secretion and translocationof Yops is triggered by direct contact between the bacteriaand the eukaryotic cell. However, secretion can be trig-gered artificially by chelating Ca

2

+

ions in the medium,which leads to a massive release of Yops to the culturesupernatant. In

Salmonella

,

Shigella

and

Yersinia

, thetype III secretion apparatus, called injectisome, is com-posed of a large dual-ring structure spanning the twobacterial membranes, resembling the flagellum basalbody. This structure is topped outside the bacterium by an

ª

600-nm-long needle (Kubori

et al

., 1998; Blocker

et al

.,2001; Hoiczyk and Blobel, 2001). The exact nature of theYops secretion signal(s) is still a matter of controversy. Ithas been demonstrated that the first 15 amino acidsor codons of YopE are sufficient to allow the secretion ofreporter proteins (Sory

et al

., 1995; Anderson andSchneewind, 1997) (Fig. 1).

Secretion of some TTS substrates requires individualchaperones, a new family collectively called type III secre-tion chaperones (Wattiau

et al

., 1996). The loss of suchchaperones affects only the secretion of the cognate sub-strate, without altering the secretion of other proteins(Wattiau

et al

., 1994). In many cases, the loss of a specificchaperone also results in reduction or absence of thesubstrate protein in the cytoplasm. There is little or nosequence similarity between the different TTS chaper-ones, but they share common properties. They are small

1184

M. F. Feldman, S. Müller, E. Wüest and G. R. Cornelis

© 2002 Blackwell Publishing Ltd,

Molecular Microbiology

,

46

, 1183–1197

(<20 kDa), acidic proteins with a C-terminal amphipathichelix. Some chaperones, including SycE, the chaperoneof YopE, form dimers (Wattiau and Cornelis, 1993; Birtalanand Ghosh, 2001). The region between the amino acids16 and 50 of YopE is enough to promote binding of thechaperone (Woestyn

et al

., 1996). In a recent work,Birtalan

et al

. (2002) solved the crystal structure of thecomplex between the N-terminal region of YopE and SycE.From this report, it appears that the interaction betweenYopE and SycE extends to amino acid 78 of YopE (Fig. 1).In

Yersinia

, effectors YopE, YopH and YopT have a dedi-cated chaperone, whereas YopP, YopO/YpkA or YopM donot seem to need any type III chaperone to be secreted.Some Yops are not effectors. They form a pore into theeukaryotic cell target and allow the translocation ofthe Yop effectors across the eukaryotic plasma membrane(Hakansson

et al

., 1996; Neyt and Cornelis, 1999). TheseYops, namely YopB and YopD, share a chaperone calledSycD (Wattiau

et al

., 1994).A number of functions have been proposed for TTS

chaperones. It has been shown that SycE bindingstabilizes YopE, preventing its intrabacterial degradation(Frithz-Lindsten

et al

., 1995). It has also been suggestedthat SycE plays a role as an accessory secretion pilot,mediating the recognition of YopE by the injectisome(Cheng and Schneewind, 1999). However, this pilotingrole for SycE is controversial, as secretion of YopE canoccur in the absence of the chaperone (Schesser

et al

.,1996; Woestyn

et al

., 1996). It has also been proposedthat SycE could play a role as a factor introducing ahierarchy among the effectors to be delivered (Boyd

et al

.,2000a), and a similar function has been suggestedrecently for SycH (Wulff-Strobel

et al

., 2002). It has beenhypothesized that binding of the chaperone to the effec-tors could constitute three-dimensional signals that mayendow a temporal order to secretion (Birtalan

et al

.,2002). The homologue of SycD in

Shigella

, IpgC, acts asa bodyguard preventing premature associations betweensecreted proteins that interact outside the bacterial cell(Menard

et al

., 1994). A similar function was proposed for

SicA from

Salmonella enterica

(Tucker and Galan, 2000).However, the role of SicA is more complex, as SicA alsointeracts with a transcriptional regulator modulating itsactivity (Darwin and Miller, 2001). Recently, it has beenproposed that the chaperone Spa15 in

Shigella

interactswith several partners (Page

et al

., 2002).The crystal structures of the complexes formed by the

chaperone-binding domains of YopE and SptP, an effectorprotein of

S. enterica

, and their cognate chaperones SycEand SicP were solved recently (Stebbins and Galan, 2001;Birtalan

et al

., 2002). Despite the fact that the proteinsshare very little sequence homology, the crystal structuresof both complexes are similar. These structures clearlyrevealed that the chaperone-binding domain is maintainedin an extended, unfolded conformation by means of thechaperone. Although no information was given in theirwork about how the unfolding of this domain affects thestructure of the remaining domains of SptP, Stebbinsand Galan (2001) suggested that TTS chaperones main-tain the respective substrates in a secretion-competentstate, compatible with secretion. However, in their paper,Birtalan

et al

. (2002) also observed that YopE complexedto SycE exhibits activity, leading to the conclusion that thechaperone action does not extend globally over the entireeffector. This observation is in agreement with a previousreport describing the interactions between

Escherichiacoli

Tir and

S. enterica

SigD effectors and their respectivechaperones CesT and SigE (Luo

et al

., 2001). However,so far, no crystal structure of any full-length effectorcomplexed to a type III chaperone has been reported.Thus, the actual role(s) of TTS chaperones remain(s)elusive.

One of the best understood translocation processes isthe transport of proteins into mitochondria (Pfanner andGeissler, 2001). In this system, the imported proteins arethreaded through the translocation machinery as linearchains, although larger structures can be tolerated by theimport channels. Some chaperones are required to main-tain proteins in an unfolded state or even to unfoldcompletely folded structures. The enzyme dihydrofolate

Fig. 1.

The different domains of YopE. Codons 1–15 constitute a signal sufficient to allow secretion of several chimeric proteins. Amino acids 15–50 are necessary and sufficient for binding of the chaperone SycE. However, the crystal structure of the complex between the N-terminal region of YopE and SycE shows that the region of YopE interacting with SycE extends to amino acid 78 (Birtalan

et al

., 2002). YopE

D

50

-

77

can be secreted without SycE, and only when this region is present is the chaper-one needed (Boyd

et al

., 2000a). Thus, the region between amino acids 50 and 77 has a negative effect on secretion. The catalytic domain of YopE extends from amino acid 90 onwards (Evdokimov

et al

., 2002).

Role of SycE in type III secretion

1185



,

46

, 1183–1197

reductase (DHFR) has been used extensively to studyprotein translocation into organelles, and its folding prop-erties are very well known (Clark and Frieden, 1999). Ithas been shown that point mutations destabilizing mouseDHFR enhance its post-translational import into mitochon-dria (Vestweber and Schatz, 1988). The binding of folateanalogues to DHFR before its import to mitochondria sta-bilizes the folded conformation, blocking its translocation

in vitro

(Eilers and Schatz, 1986). These experimentswere crucial to demonstrate that the precursors mustunfold in order to be imported into mitochondria. Folateanalogues also inhibit DHFR translocation into lysosomes(Salvador

et al

., 2000) and endoplasmic reticulum (ER)(Schlenstedt

et al

., 1990). However, in other systems,folded proteins can be translocated across membranes.Examples are transport into peroxisomes (Walton

et al

.,1995), bacterial type II secretion systems (Pugsley, 1992)and the Tat pathway in

E. coli

and thylakoid membranes(Hynds

et al

., 1998).In this work, we have studied the secretion of several

YopE–DHFR fusion proteins by the TTS system in

Y.enterocolitica

and have analysed the role of SycE inthis process. Our

in vivo

results indicate that proteinstravel through the type III channel in a partially foldedor unfolded conformation and that the interaction ofSycE with its substrate renders a protein in a secretion-competent state compatible with secretion by TTS.

Results

Mouse DHFRwt fused to the YopE secretion signal is not secreted by a multimutant strain of

Y. enterocolitica

In order to investigate how proteins travel through the typeIII channels, we have chosen mouse DHFR as a modelprotein. We have analysed the secretion of DHFR fusionproteins in the multimutant

D

HOPEMT

Y. enterocolitica

strain. This strain lacks all the effector proteins, and com-petition for delivery is reduced or eliminated (Table 1)(Boyd

et al

., 2000a). In this strain, YopN and the translo-cator Yops (YopB, YopD and LcrV), as well as other com-ponents of the TTS system such as YscP, can still besecreted. We found that a fusion protein containing thefirst 16 amino acids of YopE and the wild-type mouseDHFR (DHFRwt) encoded by the moderate-copy-numbervector pBBR1MCS-2 (Kovach

et al

., 1995) and tran-scribed from the

yopE

promoter was not secreted by

D

HOPEMT (Fig. 2A, lane 2; Fig. 3A, lane 1). This obser-vation was intriguing as the same signal under similarexperimental conditions allows the secretion of severalother reporter proteins (Sory and Cornelis, 1994; Ander-son and Schneewind, 1997). To rule out the possibility thatYopE

16

–DHFRwt was not secreted because it was fullyaggregated in the cytoplasm, we lysed bacteria by ultra-sonic treatment and separated the unbroken cells by low-

speed centrifugation. The supernatant was then ultracen-trifuged, and the resulting fractions were analysed byWestern blot. We found that YopE

16

–DHFR remained sol-uble (Fig. 2B, lane 1), which suggested that the lack ofsecretion was not caused by aggregation of the fusionproteins as a result of overexpression or a change in thesolubility of DHFR. Instead, taken together, these findingssuggested that lack of secretion is related to an intrinsicproperty of DHFRwt. Although it was not secreted,YopE

16

–DHFRwt did not obstruct the secretion channel,as YopN and the translocators were still secreted normally(Fig. 2A, lane 2). This suggests that the non-secretedYopE

16

–DHFRwt was rejected from the channel.

Fig. 2.

Secretion of YopE–DHFRwt hybrid proteins by

Y. enterocolitica

D

HOPEMT cells.A.

Y. enterocolitica

D

HOPEMT bacteria expressing YopE

16

–DHFRwt or YopE

52

–DHFRwt were grown in conditions inducing Yop secretion. The proteins secreted (S) by 5

¥

10

8

bacteria and the pellets (P) of 5

¥

10

7

bacteria were collected and analysed by SDS-PAGE and Coomassie staining. Lanes 1 and 4, control

D

HOPEMT (pNGB2); lanes 2 and 5,

D

HOPEMT (pMAF12); lanes 3 and 6,

D

HOPEMT (pMAF14). The asterisks indicate the positions of the hybrid proteins.B. Cells expressing YopE–DHFRwt were lysed and centrifuged at low speed to remove hypothetical inclusion bodies and unbroken cells. The supernatants were ultracentrifuged, and the soluble (S) and insoluble (I) fractions of this ultracentrifugation were analysed by Western blot with antibodies against DHFR. Lanes 1 and 2,

D


D


D

HOPEMT

D

SycE (pMAF14).

1186

M. F. Feldman, S. Müller, E. Wüest and G. R. Cornelis



,

46

, 1183–1197

Tab

le 1

.

Str

ains

and

pla

smid

s us

ed in

this

stu

dy.

Pla

smid

sR

elev

ant

char

acte

ristic

sR

efer

ence

pYV

der

ivat

ives

pIM

L421

PY

V40

:

yopH

D

1

-

352

yopO

D

65

-

558

yopP

23

yopE

21

yopM

23

yopT

135

.Ir

iart

e an

d C

orne

lis (

1998

)E

40 c

arry

ing

this

pla

smid

is c

urre

ntly

cal

led

D

HO

PE

MT

pIM

L422

PY

V40

:

yopH

D

1

-

352

yopO

D65

-

558

yopP

23

yop

E21

yop

M23

yop

T13

5Ir

iart

e an

d C

orne

lis (

1998

)sy

cE54

. E40

car

ryin

g th

is p

lasm

id is

cur

rent

ly c

alle

d DH

OP

EM

T D

Syc

E

Clo

nes

and

vect

ors

R38

8E

ncod

es a

pt-in

sens

itive

DH

FR

Hei

dorn

and

Val

entin

e (1

986)

pBB

R1M

CS

-2B

road

-hos

t-ra

nge,

mod

erat

e-co

py-n

umbe

r cl

onin

g ve

ctor

.K

ovac

h et

al.

(199

5)G

enB

ank

acce

ssio

n nu

mbe

r U

2375

1pX

VI2

Sou

rce

of m

ouse

DH

FR

Stu

eber

et a

l. (1

984)

pGE

M4-

DH

FR

mut

Sou

rce

of D

HF

Rm

ut (

Cys

-7Æ

Ser

, S

er-4

2ÆC

ys a

nd A

sn-4

9ÆC

ys)

Teic

hman

n et

al.

(199

6)pA

PB

26Yo

pE c

lone

d un

der Y

opE

pro

mot

erB

oyd

et a

l. (2

000b

)pN

GB

2V

ecto

r fo

r en

gine

erin

g Yo

pE16

hyb

rid p

rote

ins

unde

r Yop

E p

rom

oter

. Hig

h co

py n

umbe

rB

oyd

et a

l. (2

000a

)pN

GB

4D

HF

Rw

t cl

oned

in B

glII–

Xba

I si

tes

of p

NG

B2.

Enc

odes

Yop

E16

–DH

FR

wt

N. G

rosd

ent

and

G. R

. Cor

nelis

(un

publ

ishe

d)pM

AF

6D

HF

RD7

7 cl

oned

in B

glII–

Hin

dIII

of p

NG

B2.

Enc

odes

Yop

E16

–DH

FR

D77

Thi

s st

udy

pMA

F7

Ori C

olE

1, o

riTR

K2,

bla

, P

yopE

–SD

F10

–Yop

E52

–Bgl

II–X

baI–

Hin

dIII.

Vec

tor

for

engi

neer

ing

Yop 5

2 hy

brid

pro

tein

s un

der Y

opE

pro

mot

erT

his

stud

ypM

AF

8D

HF

Rw

t cl

oned

in B

glII–

Xba

I si

tes

of p

MA

F7.

Enc

odes

Yop

E52

–DH

FR

wt

Thi

s st

udy

pMA

F9

DH

FR

mut

clo

ned

in B

glII–

Xba

I si

tes

of p

MA

F7.

Enc

odes

Yop

E52

–DH

FR

mut

Thi

s st

udy

pMA

F10

DH

FR

D77

clon

ed in

Bgl

II–H

indI

II of

pM

AF

7. E

ncod

es Y

opE

52–D

HF

RD7

7T

his

stud

ypM

AF

11D

HF

Rm

ut c

lone

d in

Bgl

II–X

baI

site

s of

pN

GB

2. E

ncod

es Y

opE

16–D

HF

Rm

utT

his

stud

ypM

AF

12Yo

pE16

–DH

FR

wt

unde

r Yop

E p

rom

oter

clo

ned

from

pN

GB

4 in

Eco

RI–

Hin

dIII

site

s of

pB

BR

1MC

S-2

Thi

s st

udy

pMA

F13

YopE

16–D

HF

RD7

7 un

der Y

opE

pro

mot

er c

lone

d fr

om p

MA

F6

in E

coR

I–H

indI

II si

tes

of p

BB

R1M

CS

-2T

his

stud

ypM

AF

14Yo

pE52

–DH

FR

wt

unde

r Yop

E p

rom

oter

clo

ned

from

pM

AF

8 in

Eco

RI–

Hin

dIII

site

s of

pB

BR

1MC

S-2

Thi

s st

udy

pMA

F15

YopE

52–D

HF

Rm

ut u

nder

Yop

E p

rom

oter

clo

ned

from

pM

AF

9 in

Eco

RI–

Hin

dIII

site

s of

pB

BR

1MC

S-2

Thi

s st

udy

pMA

F16

YopE

52–D

HF

RD7

7 un

der Y

opE

pro

mot

er c

lone

d fr

om p

MA

F10

in E

coR

I–H

indI

II si

tes

of p

BB

R1M

CS

-2T

his

stud

ypM

AF

17Yo

pE16

–DH

FR

mut

und

er Y

opE

pro

mot

er c

lone

d fr

om p

MA

F11

in E

coR

I–H

indI

II si

tes

of p

BB

R1M

CS

-2T

his

stud

ypM

AF

29C

onta

insY

opE

17-5

2–D

HF

Rw

t in

Nde

I–X

baI

site

s of

pN

GB

2T

his

stud

ypM

AF

30Yo

pE17

-52–

DH

FR

wt

unde

r Yop

E p

rom

oter

clo

ned

from

pM

AF

29 in

Eco

RI–

Hin

dIII

site

s of

pB

BR

1MC

S-2

Thi

s st

udy

pMA

F33

Con

tain

s S

ycE

in N

coI–

Hin

dIII

pBA

D-M

yc-H

isC

. Exp

ress

es S

ycE

onl

y in

the

pre

senc

e of

ara

bino

seT

his

stud

ypM

AF

34C

onta

ins

the

full-

leng

th Y

opE

in N

deI–

Bgl

II si

tes

of p

MA

F8.

Enc

odes

Yop

EF

L–D

HF

Rw

tT

his

stud

ypM

AF

35Yo

pEF

L–D

HF

Rw

t un

der Y

opE

pro

mot

er c

lone

d fr

om p

MA

F34

in E

coR

I–H

indI

II si

tes

of p

BB

R1M

CS

-2T

his

stud

ypM

AF

38Yo

pE80

clo

ned

in N

deI–

Bgl

II si

tes

of p

MA

F8.

Enc

odes

Yop

E80

–DH

FR

wt.

Thi

s st

udy

pMA

F39

YopE

80–D

HF

Rw

t un

der Y

opE

pro

mot

er c

lone

d fr

om p

MA

F38

in E

coR

I–H

indI

II si

tes

of p

BB

R1M

CS

-2T

his

stud

ypM

AF

44D

HF

RD7

7 cl

oned

in B

glII–

Hin

dIII

of p

MA

F38

. Enc

odes

Yop

E80

–DH

FR

D77

Thi

s st

udy

pMA

F45

DH

FR

D77

clon

ed in

Bgl

II–H

indI

II of

pM

AF

34. E

ncod

es Y

opE

FL–

DH

FR

D77

Thi

s st

udy

pMA

F46

YopE

80–D

HF

RD7

7 un

der Y

opE

pro

mot

er c

lone

d fr

om p

MA

F44

in E

coR

I–H

indI

II si

tes

of p

BB

R1M

CS

-2T

his

stud

ypM

AF

47Yo

pEF

L–D

HF

RD7

7 un

der Y

opE

pro

mot

er c

lone

d fr

om p

MA

F45

in E

coR

I–H

indI

II si

tes

of p

BB

R1M

CS

-2T

his

stud

y

Role of SycE in type III secretion 1187

© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 1183–1197

A misfolded truncated form of DHFR can be secreted fused to the N-terminal secretion signal of YopE

One possible explanation for the fact that the N-terminalsecretion signal did not promote the secretion of DHFRwtis that the folding properties of the enzyme may be incom-patible with secretion by the TTS system. We reasonedthat the introduction of some mutations known to reducethe stability of DHFR could allow the secretion of theYopE16–DHFR hybrid under the same conditions. Wemade a construct containing the N-terminal secretion sig-nal of YopE and DHFRmut (Cys-7ÆSer, Ser-42ÆCys andAsn-49ÆCys), a mutant that has been used previously tostudy import of proteins into mitochondria (Vestweber andSchatz, 1988) and to investigate protein degradationinside this organelle (Teichmann et al., 1996). DHFRmutis less stable than DHFRwt and is unable to bind folateanalogues. However, the fusion protein YopE16–DHFRmutwas not secreted by Y. enterocolitica either (Fig. 3A,lane 2).

We then tested the secretion of a truncated form ofDHFR lacking the last 77 amino acids. This DHFRD77mutant form has been used to show that the Tat secretionsystem can translocate misfolded proteins through thethylakoid membrane (Hynds et al., 1998). In contrast tothe first two hybrids, DHFRD77 fused to the first 16 aminoacids of YopE was secreted efficiently (Fig. 3A, lane 3) bythe DHOPEMT strain.

The secretion experiments described so far were alsocarried out in a wild-type Yersinia strain and in an yscNmutant strain. YscN is the ATPase of the Ysc system andis absolutely required for secretion (Woestyn et al., 1994).The wild-type strain gave the same results as DHOPEMT,whereas the yscN mutant strain did not secrete any of theDHFR hybrid proteins, indicating that secretion occurredvia the Ysc TTS system (data not shown).

Intrabacterial stability of the DHFR mutants

In order to understand the different behaviour of themutants and to check whether there was any correla-tion between the degree of unfolding and the rate ofsecretion, we compared the intrabacterial stability ofYopE16–DHFRwt, YopE16–DHFRmut and YopE16–DHFRD77 (Fig. 4). Y. enterocolitica cells were grown inbrain–heart infusion (BHI) medium containing 2.5 mMCa2+ to keep the channel closed, and chloramphenicolwas added to the medium in order to stop protein synthe-sis. Aliquots of the cultures were taken at different timepoints, and the bacterial pellets were analysed by Westernblotting. We found that YopE16–DHFRwt was stable for>1 h at 37∞C, suggesting that the protein was folded andtherefore could resist degradation by bacterial proteases.YopE16–DHFRD77 was the most unstable of the threehybrid proteins with a half-life of <5 min, and YopE16–

Fig. 3. Effect of DHFR mutations and of SycE on secretion of YopE–DHFR fusion proteins.A. Bacterial cells expressing the indicated YopE–DHFR fusion pro-teins were grown in conditions inducing Yop secretion. Cultures were centrifuged, and the pellets were treated with proteinase K to destroy protein aggregates that could co-sediment with the bacterial cells. Culture supernatants were precipitated by TCA, and both pellets and supernatants were analysed by Western blot with antibodies against DHFR. Top: bacterial pellets of strain DHOPEMT. Bottom: superna-tants from strain DHOPEMT. Lanes: 1, DHOPEMT (pMAF12); 2, DHOPEMT (pMAF17); 3, DHOPEMT (pMAF13); 4, DHOPEMT (pMAF14); 5, DHOPEMT (pMAF15); 6, DHOPEMT (pMAF16).B. Dependence on arabinose of the expression of SycE from pMAF33 (see Table 1) in DHOPEMT DSycE cells. The expression of SycE was analysed by Western blot using anti-SycE antibodies. SycE was only detected upon induction of expression with 0.2% arabinose.C. Secretion of YopE52–DHFR fusion proteins in the DHOPEMT DSycE (pMAF33) strain. Only the culture supernatants are shown. Lanes 1 and 2, DHOPEMT DSycE (pMAF33) (pMAF14); lanes 3 and 4, DHOPEMT DSycE (pMAF33) pMAF15; lanes 5 and 6, DHOPEMT DSycE (pMAF33) (pMAF16).

1188 M. F. Feldman, S. Müller, E. Wüest and G. R. Cornelis


DHFRmut had an intermediate stability with a half-life ofª 12–15 min. This result indicated that the folding ofYopE16–DHFRD77 was more drastically affected than thefolding of YopE16–DHFRmut. Thus, increased destabiliza-tion of the DHFR domain allowed secretion of the fusionproteins via the TTS system, suggesting that the N-termi-nal secretion signal of YopE can only promote the secre-tion of severely unfolded DHFR domains. The fact thatYopE16–DHFRD77 could be secreted efficiently (Fig. 3A)in spite of its very short half-life also indicates that thesecretion process is very rapid and efficient.

Effect of SycE on secretion of hybrid YopE–DHFR proteins by the Ysc TTS system in Y. enterocolitica

As we could not observe secretion of YopE16–DHFRwt, wedecided to test whether SycE could favour secretion of aYopE–DHFRwt fusion protein. We thus engineered aYopE–DHFRwt fusion containing 52 residues of YopE, i.e.the minimal region needed to bind SycE (Woestyn et al.,1996). When the analysis was performed in DHOPEMT,in which SycE is normally expressed, we found thatYopE52–DHFRwt was secreted to the extracellularmedium (Fig. 2A, lane 3; Fig. 3A, lane 4). YopE52–DHFR-mut and YopE52–DHFRD77 were also secreted well inthese conditions (Fig. 3A, lanes 5 and 6). To confirm theseresults further, we transformed the DHOPEMT DSycEstrain with a plasmid expressing sycE under the control

of the E. coli arabinose-inducible operon promoter (PBAD).In this strain, there is no detectable SycE unless arabi-nose is added to the medium (Fig. 3B). We found thatYopE52–DHFRwt was secreted strictly upon addition ofarabinose and consequent SycE induction (Fig. 3C, lanes1 and 2). YopE52–DHFRmut was secreted well in the pres-ence of the inducer, and some protein was still secretedin the absence of arabinose (Fig. 3C, lanes 3 and 4).YopE52–DHFRD77 was secreted well in the presence andabsence of SycE (Fig. 3C, lanes 5 and 6).

The lack of secretion of YopE52–DHFRwt in the absenceof SycE was not caused by aggregation of the proteininside the bacterial cells, as the protein can be detectedin the soluble fractions of the cells after ultracentrifugation(Fig. 2B). To rule out the possibility that YopE52–DHFRwtwas not secreted because of dimerization or multimeriza-tion of DHFR or the formation of soluble complexes withother cytosolic proteins in the absence of SycE, we loadedextracts of DHOPEMT DSycE cells expressing the hybridYopE52–DHFRwt in a Superdex 75 column. Most of theprotein eluted in the volume corresponding to the mole-cular weight expected for monomeric YopE52–DHFRwt(data not shown), indicating that SycE did not promotesecretion of YopE52–DHFRwt by avoiding non-productiveDHFR interactions.

To ensure that the SycE-binding domain did not pro-mote secretion by itself acting as a second secretionsignal, we tested the secretion of YopE17-52–DHFRwt, afusion protein that lacks the N-terminal secretion signal ofYopE. This fusion protein was not secreted (Fig. 5A) and,hence, only the hybrid containing DHFRwt fused to boththe secretion signal and the minimal SycE-binding domainwas secreted.

We carried out overlay experiments using an E. coliextract containing SycE to show that all the fusion proteinscontaining the minimal SycE-binding domain (amino acids17–52) were able to bind SycE (Fig. 5B). In addition, theproteins lacking this region did not bind detectableamounts of SycE (Fig. 5B). Therefore, our results clearlyconfirmed that amino acids 17–52 of YopE are enough tobind SycE and constitute a functional minimal bindingdomain.

Taken together, these experiments demonstrate thatSycE binding to YopE52–DHFRwt promotes the secretionof the DHFRwt domain. The secretion absolutely requiresthe N-terminal secretion signal, the minimal SycE-bindingdomain and a functional SycE present in the cell. Therequirement for SycE was less strict for DHFRmut, andDHFRD77 did not require SycE for secretion.

Effect of aminopterin on the secretion of YopE52–DHFR

As the interaction between SycE and YopE52–DHFRwt

Fig. 4. Intrabacterial stability of YopE16–DHFR chimeric proteins. DHOPEMT (pMAF12), DHOPEMT (pMAF17) and DHOPEMT (pMAF13) Y. enterocolitica cells (see Table 1) were grown at 37∞C in presence of 5 mM Ca2+. At time 0, chloramphenicol was added in order to stop new protein synthesis. Aliquots were taken at different time intervals, and the amount of proteins was detected by Western blot and quantified by densitometric analysis.



provides a secretion-competent fusion protein to the Yscmachinery, we wanted to know whether this state was theresult of active unfolding of completely folded structuresor, alternatively, the consequence of preventing folding ofYopE52–DHFRwt. Methotrexate (mtx), trimethoprim (tmp)and aminopterin (apt) are folate analogues that bind tobacterial and eukaryotic DHFR. When mtx or apt bind toDHFR, the protein is stabilized in a permanently foldedconformation, and this stabilization is strong enough formtx and apt to block the unfolding of DHFR and, accord-ingly, its import into mitochondria in vitro (Eilers andSchatz, 1986). The blocking of translocation of DHFR intomitochondria by mtx has also been observed in vivo inyeast (Wienhues et al., 1991). We thus wanted to testwhether folate analogues can block the secretion of

YopE52–DHFRwt. Mtx could not be used because Y.enterocolitica pumps it out of the cells (Kopytek et al.,2000). Apt, which has the same affinity for bacterial andmammalian DHFR as mtx (Sasso et al., 1994) at a con-centration of 1 mM, inhibits growth of Y. enterocolitica,implying that the drug enters into the cells, reaching anintracellular concentration sufficient to inhibit the bacterialDHFR. Thus, apt could be used provided the bacterium isprotected from the deleterious effect of apt on its ownDHFR, by supplying an apt-insensitive DHFR. To achievethis, we transformed DHOPEMT with tmp resistance plas-mid R388, which encodes an apt-insensitive DHFR(Narayana et al., 1995). We then analysed secretion of theYopE52–DHFRwt hybrid protein by HOPEMT (pMAF14)(R388) and found that the addition of 1 mM apt to the cellsdid not prevent secretion of the YopE52–DHFRwt fusion(Fig. 6). This result favours the hypothesis that SycE inter-acts with DHFRwt preventing the folding of YopE52–DHFRwt rather than promoting its unfolding.

Hybrids containing the 80 N-terminal residues of YopE (YopE80) or full-length YopE (YopEFL ) and DHFRwt are not secreted and obstruct the channel

We showed that the activity of SycE promotes the secre-tion of the DHFRwt domain if it is fused to the N-terminal52 amino acids of YopE. As mentioned before, in the

Fig. 5. A. The SycE-binding domain is necessary but not sufficient for secretion of YopE–DHFR hybrids. DHOPEMT cells carrying plas-mids pMAF12 (lanes 1 and 4), pMAF14 (lanes 2 and 5) and pMAF30 (lanes 3 and 6) (see Table 1) were grown in conditions inducing Yop secretion. The supernatants of 5 ¥ 108 cells (S) and total 5 ¥ 107 cells (P) were analysed by Western blot using anti-DHFR antibodies.B. SycE-binding assay. DHOPEMT cells expressing different YopE–DHFR fusions were grown in conditions promoting Yops induction. The cultures were centrifuged, and the bacterial proteins were sep-arated by SDS-PAGE, transferred to a nitrocellulose membrane and incubated with an E. coli extract overproducing SycE. Binding of SycE was detected using anti-SycE antibodies. The band corresponding to SycE protein is indicated (arrow).

Fig. 6. Effect of aminopterin on secretion of YopE52–DHFRwt. Cells were incubated in conditions inducing Yops secretion in the presence and absence of 1 mM aminopterin (see Experimental procedures). The proteins secreted by 5 ¥ 109 bacteria were precipitated and anal-ysed by SDS-PAGE and Coomassie staining.



crystal of the complex between SycE and the N-terminalregion of YopE, SycE interacts with the first 78 amino acidsof YopE (Birtalan et al., 2002) (Fig. 1). We decided totest whether SycE binding to a hybrid protein containingthe complete region covered by the chaperone is alsoable to promote secretion of the DHFRwt domain. Wefound that, unlike YopE52–DHFRwt, YopE80–DHFRwt wasnot secreted. On the contrary, YopE80–DHFRD77 wassecreted efficiently (Fig. 7A). We also tested the secretionof hybrid proteins containing full-length YopE (YopEFL) andfound that the hybrid YopEFL–DHFRwt was not secretedeither. However, YopEFL–DHFRD77 was detected in goodamounts in the culture supernatants (Fig. 7A). There-fore, all the hybrids between YopE and DHFRD77 weresecreted regardless of the length of the YopE sequencecontained in the chimeric protein and irrespective ofthe presence or absence of SycE. In contrast, onlythe YopE52–DHFRwt hybrid could be secreted, and thissecretion was SycE dependent. In the YopEFL–DHFRwtand YopE80–DHFRwt hybrids, the longer YopE domainsbehave as spacers. As a result, SycE cannot interact withDHFRwt and, consequently, the effect of the binding ofSycE on these proteins is lost. This result suggests that,in the case of YopE52–DHFRwt, SycE prevents DHFRfolding by overlapping the protein physically.

We analysed by SDS-PAGE and Coomassie staining

the supernatants of the DHOPEMT strain expressingYopEFL–DHFRwt and YopE80–DHFRwt (Fig. 7B). Wefound that the expression of YopEFL–DHFRwt almost com-pletely abrogated the secretion of most of YopN and thetranslocator Yops. Only a few proteins were still secretedin these conditions. This effect was less marked in thecase of YopE80–DHFRwt than in the case of YopEFL–DHFRwt (Fig. 7B). These results suggest that, whenYopEFL–DHFRwt is engaged in the secretion channel bythe YopE moiety, it blocks the secretion of other Yops byobstruction of the channel through the tightly folded DHFRdomain in an irreversible way. This is additionally sup-ported by the observation that the amounts of the YopEFL–DHFRwt and YopE80–DHFRwt hybrids in the bacterial

Fig. 7. YopEFL–DHFRwt and YopE80–DHFRwt are not secreted and block secretion of the other Yops in a SycE-dependent fashion. The secretion of hybrids containing either YopEFL or YopE80 fused to DHFRwt or DHFRD77 was analysed in the presence or absence of SycE.A. Western blot analysis of intracellular and secreted YopE–DHFR hybrids by DHOPEMT cells transformed with pMAF39, pMAF46, pMAF35 and pMAF47 (see Table 1). The detection was made with antibodies anti-YopE. The asterisk points out truncated secreted pro-teins, most probably resulting from degradation of YopEFL–DHFR77.B. Analysis by SDS-PAGE and Coomassie staining of the proteins secreted by DHOPEMT transformed with plasmids pMAF35 (lane 3) and pMAF39 (lane 4; see Table 1). DHOPEMT (pMAF14) (lane 1) and DHOPEMT (pMAF16) (lane 2) are presented for comparison. Note the absence or reduction of most of the bands corresponding to the remaining Yops. The asterisks point out two minor bands correspond-ing to proteins still secreted in these conditions.C. Analysis similar to that presented in (B) but in the DHOPEMT DSycE strain transformed with pMAF33 that expresses SycE upon induction with arabinose (see Table 1 and Fig. 3B). Cultures of DHOPEMT DSycE (pMAF33) were split into two cultures, and arabi-nose was added to one of them. Note that expression of YopEFL–DHFRwt absolutely abrogates secretion of the Yops normally secreted by the host strain only when production of SycE is induced (compare lanes 4 and 9). This effect is less marked in the case of YopE80–DHFRwt (compare lanes 5 and 10). The secretion patterns of DHOPEMT DSycE (pMAF33) expressing YopE52–DHFRwt, YopE52–DHFRmut and YopE52–DHFRD77 are presented for comparison.D. YopE80–DHFRwt and YopEFL–DHFRwt are unstable in the absence of SycE. Proteins from 5 ¥ 108 total cells grown in the presence of absence of arabinose were analysed by Western blot using antibod-ies anti-YopE and DHFR. Left, DHOPEMT DSycE (pMAF33) (pMAF35); right, DHOPEMT DSycE (pMAF33) (pMAF39).



cells were considerably lower than the amounts ofYopEFL–DHFRD77 and YopE80–DHFRD77 (Fig. 7A). Thisis probably the result of feedback inhibition on transcrip-tion, a phenomenon commonly observed when Yops areproduced with the channel closed by the presence of Ca2+

or by mutation (Cornelis et al., 1987).We then expressed these fusion proteins in the

DHOPEMT DSycE (pMAF33) strain, which expressesSycE from an arabinose-inducible promoter (see Table 1).We found that, in the absence of arabinose, YopEFL–DHFRwt and YopE80–DHFRwt did not prevent secretion ofthe remaining Yops, implying that jamming of the injecti-some was dependent on SycE (Fig. 7C). It has beendemonstrated several years ago that, in the absence ofSycE, YopE aggregates and is degraded (Wattiau andCornelis, 1993; Frithz-Lindsten et al., 1995). By analogy,we suspected that the SycE-dependent obstruction ofthe injectisome by YopEFL–DHFRwt and YopE80–DHFRwtresulted from instability of these proteins in the absenceof the chaperone. We thus analysed the intrabacterialproteins of DHOPEMT DSycE (pMAF33) cells expressingeither YopEFL–DHFRwt or YopE80–DHFRwt in the pre-sence or absence of arabinose. In both cases, anti-YopEantibodies detected considerably less hybrid proteins inthe absence of arabinose than in the presence ofthe inducer (Fig. 7D). However, anti-DHFR antibodiesdetected a band of lower molecular weight in the lanescorresponding to both hybrid proteins produced in theabsence of arabinose (Fig. 7D). This band is most prob-ably the degradation-resistant DHFRwt moiety resultingfrom proteolytic digestion of YopEFL–DHFRwt andYopE80–DHFRwt. These results indicate that, in the absence ofSycE, both proteins are unstable and explain why thesehybrids do not block the channel in the absence of SycE.

Discussion

In this study, we addressed the questions of the foldingrequirements of TTS substrates in order to be secreted,and of the role of the TTS-specific chaperones in thiscontext. We investigated the secretion of various forms ofa reporter protein fused to parts of YopE by the Ysc TTSsystem of Y. enterocolitica and monitored the impact ofthe presence or absence of the SycE chaperone.

DHFR as a model protein to study TTS

It has been shown previously that the signal contained inthe first 15 amino acids of YopE is sufficient to allowsecretion of proteins such as alkaline phosphatase(PhoA) (Michiels and Cornelis, 1991), neomycin phos-phatase (Npt) (Anderson and Schneewind, 1997),calmodulin-dependent adenylate cyclase of Bordetellapertussis (Cya) (Sory and Cornelis, 1994) and the frag-

ment A of the diphtheria toxin (Boyd et al., 2000b).With the exception of Npt, these proteins are intrinsicallysecreted proteins, endowed with properties for transloca-tion across membranes. In the present study, we havechosen to use DHFR as a reporter because DHFR is nota naturally secreted protein but has been widely used tostudy protein translocation to organelles, and its foldingproperties as well as that of some mutants have beendescribed (Vestweber and Schatz, 1988; Hynds et al.,1998).

We have analysed the secretion of hybrids containing16 (the N-terminal secretion signal), 52 (the minimal bind-ing domain) and 80 (the complete binding domain) aminoacids of YopE or full-length YopE with wild-type DHFRand two DHFR mutants with altered folding properties(Table 2). YopE16–DHFRwt, YopE80–DHFRwt and YopEFL–DHFRwt were not secreted. YopE52–DHFRwt was the onlyhybrid protein containing DHFRwt that was secreted,and its secretion was absolutely dependent on SycE.YopE80–DHFRwt and YopEFL–DHFRwt were not only notsecreted, but they also jammed the channel in an SycE-dependent fashion. There was an excellent inverse corre-lation between the stability of the DHFR moiety and thecapacity of the hybrid protein to be secreted. Hybridscontaining DHFRD77, the mutant whose folding proper-ties are the most drastically affected, could be secreted inall the conditions tested.

YopE–DHFR hybrids travel unfolded through the TTS channels

Our interpretation of these data is that, in order to besecreted by the TTS system, DHFR may not be fullyfolded. This is in perfect agreement with a recentlypublished report by Lee and Schneewind (2002), whoshowed that YopE15–DHFRwt and YopEFL–DHFRwt arenot secreted. These authors also showed that YopE fused

Table 2. Secretion and channel-blocking properties of YopE–DHFRhybrid proteins in the presence and absence of SycE.

Hybrid protein

Secretion Channel jamming

+SycE –SycE +SycE –SycE

YopE16–DHFRwt – – – –YopE16–DHFRmut – – – –YopE16–DHFRD77 + + – –YopE52–DHFRwt + – – –YopE52–DHFRmut + + – –YopE52–DHFRD77 + + – –YopE80–DHFRwt – – + –YopE80–DHFRD77 + NT – NTYopEFL–DHFRwt – – + –YopEFL–DHFRD77 + NT – NTYopE17-52–DHFRwt – NT – NT

NT, Not tested.



to wild-type ubiquitin cannot be secreted by the Ysc sys-tem of Y. enterocolitica, but a similar hybrid containing afolding mutant of ubiquitin is secreted. Both reports sup-port the idea that tightly folded protein domains cannot besecreted by TTS systems. The minimal diameter of foldedDHFR is ª 30 Å (Oefner et al., 1988). The size proposedfor type III channels is ª 20–30 Å (Kubori et al., 1998;Blocker et al., 2001). Therefore, although it is possible thatthe channel is too narrow to allow the secretion of fullyfolded proteins, the finding that YopE–DHFR hybrids travelthrough the injectisomes in an unfolded or partially foldedconformation is not trivial.

SycE keeps YopE52–DHFRwt unfolded

We have concluded that the DHFRwt domain must beunfolded or partially unfolded to be secreted. Theobservation that YopE52–DHFRwt could be secreted oncondition that SycE was present implies that SycE keepsDHFRwt in a secretion-competent conformation, probablyby preventing its folding. The idea that a TTS chaperonemaintains its partner in a partially unfolded state hasalready been proposed. Based on the crystal structure ofthe chaperone binding site of the Salmonella effector Sptcomplexed with its chaperone SicP, Stebbins and Galan(2001) have proposed that TTS chaperones maintain theirsubstrates in a secretion-competent state, allowing theeffector proteins to travel through the injectisome in anunfolded or partially folded manner.

Although YopE52–DHFR was secreted in the presenceof SycE, YopEFL–DHFRwt was not. Thus, in order to exertits role, SycE must bind close to the domain that it needsto assist. The crystal structure of the complex YopE–SycEshows that SycE covers 78 residues of YopE rather than58 (Birtalan et al., 2002). We found that YopE80–DHFRwtwas not secreted (Fig. 7A). In YopE80–DHFRwt andYopEFL–DHFRwt, the longer YopE domains behaved asspacers, and it seems that the distance between theSycE-binding domain and DHFRwt is too long to allowinteraction between SycE and the DHFRwt domain.This result suggests that SycE binding to YopE52–DHFRwt prevents DHFR folding by overlapping theprotein physically.

‘Secretion-competent’ versus ‘simultaneous unfolding secretion’: two models for protein secretion by the TTS system

These results strongly support a model in which theTTS substrates are kept in an unfolded or partially foldedconformation by means of their interaction with the TTSchaperones. This model is presented in Fig. 8. Probably,the chaperone binds to its cognate protein in a co-translational event, preventing the folding of its partner

(Fig. 8A, steps a and b). This ‘secretion-competent’ con-formation hypothesis (Stebbins and Galan, 2001) resem-bles the Sec system, and the function of SycE could berelated to the function of SecB, a chaperone involved inthe secretion of some proteins across the cytoplasmicmembrane by the Sec pathway (Driessen, 2001). Thefinding that aminopterin does not inhibit secretion ofYopE52–DHFRwt also sustains this model.

The alternative model would be that TSS substrate pro-teins first fold and then unfold in order to be secreted(Fig. 8B). As SycE does not contain any ATP-bindingdomain, it is highly improbable that the unfolding step canbe carried out by its direct activity. One possibility is that,in a ‘simultaneous unfolding secretion’ model, the unfold-ing step is made when the N-terminal region of the proteinis engaged in the secretion machinery. In this case, acomponent of the secretion machinery itself, such asYscN, could provide the energy input required for theunfolding process (step g). If the secretion machineryitself is responsible for the unfolding of the tightly foldedDHFRwt domain, the distance between SycE andDHFRwt should not affect secretion. The lack of secretionof YopE80–DHFRwt and YopEFL–DHFRwt is contrary to thehypothesis of ‘simultaneous unfolding secretion’ and sug-gests instead that, in order to be secreted, the DHFRwtdomain has to be presented to the secretion apparatus inan unfolded or partially folded conformation. Alternatively,the binding of SycE to its substrate in a folded conforma-tion (step f) could recruit other proteins that might beresponsible for the unfolding step (step h). Nevertheless,our experiments support the role of SycE as an anti-folding factor that keeps the hybrid protein in asecretion-competent state, preventing the folding ofYopE52–DHFRwt rather than promoting its unfolding,

Does this apply to YopE?

Can this conclusion be transposed to YopE itself? Therecent study by Birtalan and Gosh (2001) of the complexYopE–SycE suggests that the situation of YopE could bedifferent (Birtalan et al. 2002). In that study, GST–YopEwas co-expressed with SycE in E. coli, the complex GST–YopE–SycE was purified, and it was observed that itretained the GTPase-activating (GAP) activity of YopE.This suggests that SycE does not prevent folding of YopE.However, one cannot rule out the possibility that YopE isstill maintained unfolded by SycE in Yersinia. Some cyto-solic factor(s) absent from E. coli could be necessary tostabilize the YopE–SycE complex in the secretion-compe-tent conformation (Fig. 8, step d). In this scenario, thebinding of SycE to the N-terminus of the cognate proteinmight create a signal that recruits one or more solublefactors that help SycE to keep the secreted protein in asecretion-compatible conformation. However, this is only



speculation, and our results do not prove that SycE workson YopE as it acts on YopE52–DHFRwt.

Birtalan and Ghosh (2001) suggested that SycE couldform a three-dimensional signal powering recognition bythe secretion apparatus. Thus, the structure brings somesupport to our previous hypothesis that the chaperoneintroduces some kind of hierarchy for secretion of theeffector in order to optimize the attack on the cell (Boydet al., 2000a). Although we still find this hierarchy hypoth-esis attractive, the new data from Birtalan and Ghosh(2001) also show that SycE covers the region betweenamino acids 50 and 77 of YopE. We have found previously

that YopED50-77 is perfectly secreted in the absence ofthe chaperone, and we postulated that the 50–77 regionexerts an ‘inhibitory’ effect on the secretion of YopE(Fig. 1) (Boyd et al., 2000a). Thus, the structure data givesome support to the hypothesis that one of the roles forSycE on YopE is to hide the 50–77 region. Surprisingly,this domain is not necessary for the GAP activity of YopE(Evdokimov et al., 2002). Obviously, its role cannot be toblock secretion. Thus, we suggest that it plays an addi-tional role inside the target cell. A comparative study anal-ysing the toxic effects of YopE and YopED50-77 would helpto clarify the actual role of this inhibitory region.

Fig. 8. Hypothetical models of protein secretion by TTS systems.A. In this model, the protein is never folded on its way to the secretion machinery. The chaperone binds to its substrate before the protein is fully synthesized (step a), and its binding keeps the protein in a partially folded or unfolded conformation (b), competent for secretion (c). Alternatively, other soluble factors are needed to stabilize the complex in this conformation (d).B. In this model, the TTS substrate proteins first fold and then unfold in order to be secreted. Folded proteins in the cytoplasm (e) can bind the chaperone (f). The binding of the chaperone creates an additional three-dimensional signal that is recognized by the secretion machinery, which unfolds the protein simultaneously as secretion progresses (g). Alternatively, the signal is recognized by a different chaperone that unfolds the proteins in order to allow secretion (h).Our experiments support the model presented in (A).



Rejection or jamming?

We found that YopEFL–DHFRwt not only was not secreted,but also completely blocked the injectisome (Fig. 7). Theobservation that YopEFL–DHFRwt obstructs the channelseems to be in apparent contradiction to the report by Leeand Schneewind (2002). However, in our experiments,we analysed all the proteins secreted by DHOPEMTstrains expressing YopE80–DHFRwt and YopEFL–DHFRwt,whereas in that report, only the secretion of the poorlycharacterized protein YopR is presented. We detected twoproteins that were still secreted even with the channelblocked, and one of them has the size of YopR (Fig. 7B).However, we did not advance in the identification of thesebands.

The jamming of the channel was less marked in thecase of YopE80–DHFRwt than in the case of YopEFL–DHFRwt. In contrast, although YopE52–DHFRwt wasstable in the absence of SycE (Fig. 2B) and not secreted,the channel was not jammed (Fig. 7C). YopE16–DHFRwt,which was also not secreted, did not block the channel(Table 2). This suggests that the Ysc machine rejectsfolded proteins if they are not deeply engaged into theinjectisome. Thus, blocking occurs only when a hybrid isallowed to progress into the injectisome until it reaches astep in which secretion cannot proceed because of theDHFRwt domain. This is irreversible, and the protein can-not be rejected from the secretion machinery.

Interestingly, the blockade of Yop secretion by YopE80–DHFRwt and YopEFL–DHFRwt was strictly dependent onthe presence of SycE. This is explained by the findingthat, in the absence of SycE, both hybrids are unstable(Fig. 7D). This is in perfect agreement with previousobservations showing that SycE prevents YopE aggrega-tion and stabilizes YopE inside the bacteria (Frithz-Lindsten et al., 1995). Our results suggest that the pres-ence of the 50–77 ‘inhibitory region’ is responsible forthe aggregation and consequent degradation of theprotein.

In conclusion, our experiments show that proteins travelin an unfolded or partially folded conformation through theTTS channels. We propose that SycE could have at leastthree distinct roles. By binding to the region 50–77, SycEavoids aggregation of YopE. At the same time, SycE maykeep its substrate in a secretion-competent state. Finally,it may also contribute to establishing a temporal hierarchyon secretion. However, this does not exclude differentroles for TTS chaperones, and the roles proposed forSycE do not necessarily apply to other chaperones.Indeed, the family of the type III chaperones is expandingconsiderably. The more proteins are being studied, themore interactions are being described, showing the com-plexity of the type III nanodevice.

Experimental procedures

Bacterial strains and growth conditions

This work was carried out with Y. enterocolitica MRS40multiknock-out derivatives DHOPEMT and DHOPEMT DSycE(Iriarte and Cornelis, 1998) (see Table 1). Bacteria weregrown routinely in tryptic soy broth (TSB; Oxoid) and platedon tryptic soy agar (TSA). For in vitro Yops secretion experi-ments, Y. enterocolitica cells were precultured overnight inBHI medium. Bacteria were diluted to an OD of 0.1 and grownwith shaking at room temperature in the same medium sup-plemented with 4 mg ml-1 glucose, 20 mM MgCl2 and 20 mMsodium oxalate (BHI-Ox). After 2 h, the cultures were trans-ferred to a 37∞C water bath and incubated for 4 h withshaking. When necessary, arabinose was added to a con-centration of 0.2% at the moment of the transfer to 37∞C.Selective agents were used at the following concentrations:ampicillin (200 mg ml-1), kanamycin (50 mg ml-1), nalidixicacid (35 mg ml-1) and sodium arsenite (0.4 mM). Transforma-tion of Yersinia cells was carried out by electroporation in amicropulser (Bio-Rad) according to the manufacturer'sinstructions.

Construction of plasmids

The full list of plasmids used in this study is given in Table 1.Plasmid pNGB4 (YopE16–DHFRwt) was constructed as fol-lows. The mouse DHFR gene was amplified by polymerasechain reaction (PCR) with oligonucleotides MIPA640 (ATAGATCTCGACCATTGAACTGC) and MIPA641 (GTTCTAGATTAGTCTTTCTTCTCGT) using pXIV2 as a template(Stueber et al., 1984). The oligonucleotides were designed toincorporate a BglII restriction site into the 5¢ end of the pro-duct and an XbaI site into the 3¢ end. Sites in the oligonucle-otides are underlined. The PCR product was digested withBglII and XbaI and cloned in the same sites in pNGB2 (Boydet al., 2000b). To obtain plasmid pMAF11 (YopE16–DHFR-mut), the same strategy was used, but pGEM4–DHFRmut(Teichmann et al., 1996) containing three point mutations(Cys-7ÆSer, Ser-42ÆCys and Asn-49ÆCys) was used as atemplate. To construct pMAF6 (YopE16–DHFRD77), theDHFR gene lacking the nucleotides coding for the last 77amino acids was amplified with oligonucleotides MIPA640and MIPA1035 (AAAAGCTTTCATACTTTACTTGCCAATTCC), which contains a HindIII site. The DNA fragmentobtained was cloned in the corresponding sites of pNGB2.

In order to express the hybrids containing the SycE-bindingdomain of YopE, the vector pMAF7 was constructed as fol-lows. An inverse PCR was made using primers MIPA1044and MIPA1045 and plasmid pAPB26 as a template. (Boydet al., 2000a). Primer MIPA1044 (AAAGATCTATCTAGACAATTACTGTCATTGATGGTA), hybridizing downstreamfrom yopE, contains BglII and XbaI sites. MIPA1045 (AAAGATCTACCCTGAGGGCTTTCAGTGCG) contains a BglIIsite followed by codons 52–46. The DNA fragment amplified,containing yopE truncated at codon 52, was digested withBglII and self-ligated. To construct the hybrids YopE52–DHFRwt, YopE52–DHFRmut and YopE52–DHFRD77, eachDHFR gene was amplified as described before and cloned



in the corresponding sites of pMAF7 to obtain pMAF8(YopE52–DHFRwt), pMAF9 (YopE52–DHFRmut) and pMAF10(YopE52–DHFRD77). To obtain a fusion expressing YopE17-52–DHFRwt, a PCR was made with primers MIPA1176 (AAA-CATATGTCAGGATCTAGCAGCGTAG) and MIPA641 usingpMAF8 as template. Primer MIPA1176 contains an NdeI siteand hybridizes downstream of codon 17 of YopE. The ampli-fied fragment was inserted in NdeI and XbaI sites of pNGB2to obtain pMAF29. To express a fusion protein between thefull-length YopE and DHFRwt, the entire yopE gene withoutthe stop codon was amplified with oligos MIPA1127(GGGAATAAATACATATGAAAATATCATC), containing anNdeI site, and MIPA9717 (AAAGATCTCATCAATGACAGTAATTG) possessing a BglII site. The amplified fragment wasused to replace the region coding for YopE52 in pMAF8.

In order to express YopE80–DHFRwt, plasmid pMAF38 wasconstructed as follows. The DNA region coding for the first80 amino acids of YopE was amplified with oligos MIPA1127and MF10 (AAAGATCTCTCCGAGAACATGCGTTGG) andcloned in NdeI and BglII sites replacing the region codingforYopE52 in pMAF8. Plasmid pMAF44, expressing YopE80–DHFRD77, was derived from pMAF38 replacing the regioncoding for DHFRwt by the truncated DHFRD77 gene, whichwas amplified as before and cloned in BglII and HindIII sitesof pMAF44. Plasmid pMAF45, expressing YopEFL–DHFRD77,was derived from pMAF34 with the same cloning strategy.

All the hybrids described above were subsequently sub-cloned in the moderate-copy-number vector pBBRMCS-2(Kovach et al., 1995). For this purpose, the EcoRI–HindIIIfragments containing the yopE promoter and the YopE–DHFR hybrid genes were excised from the high-copy-numberplasmids described above and cloned in the same sitesof pBBRMCS-2 in the opposite orientation to the lac pro-moter. The plasmids obtained were named pMAF12 (YopE16–DHFRwt), pMAF17 (YopE16–DHFRmut), pMAF13 (YopE16–DHFRD77), pMAF14 (YopE52–DHFRwt), pMAF15(YopE52–DHFRmut), pMAF16 (YopE52–DHFRD77), pMAF30(YopE17-52–DHFRwt), pMAF35 (YopEFL–DHFRwt), pMAF39(YopE80–DHFRwt), pMAF45 (YopEFL–DHFRD77) andpMAF47 (YopE80–DHFRD77 (see Table 1).

pMAF33 expressing sycE under the control of the PBAD

promoter was constructed inserting the sycE gene amplifiedwith primers MIPA2001 (AACCATGGTGTATTCATTTGAACAAGC) containing a NcoI site and MIPA2002 (AAAAGCTTTCAACTAAATGACCGTG), which has an HindIII site, inthe corresponding sites of pBAD/Myc-HisC (Invitrogen).

Both strands of each construct were sequenced using thesequencing kit from Beckman (CEQ DTCS no. 608 000) andan automated sequencer (Beckman). PCR for cloning pur-poses was made using Pwo DNA polymerase (Roche). Forthe inverse PCR, the Expand long template kit was used(Roche).

SDS-PAGE and Western blotting

Proteins were prepared from culture supernatants and anal-ysed by SDS-PAGE and Western blotting as described byCornelis et al. (1987), except that 10% TCA was used toprecipitate the proteins. When intrabacterial protein contentswere analysed, the cells were treated with proteinase K with

a final concentration of 500 mg ml-1 for 30 min at 37∞C inorder to digest protein aggregates that co-sedimented withthe bacterial cells.

Test of stability of DHFR mutants

Yersinia enterocolitica HOPEMT cells transformed with differ-ent DHFR constructs were grown overnight at room temper-ature in BHI. Bacteria were diluted in BHI supplemented with2.5 mM Ca2+ (BHI-Ca2+) and shifted to 37∞C. After 1 h incu-bation, chloramphenicol was added to the bacterial culture toa final concentration of 50 mg ml-1 and 1 ml samples weretaken at time points 0, 5, 10, 30 and 60 min of incubation.The samples were immediately centrifuged, and bacterialpellets were resuspended in 100 ml of sample buffer andboiled for 15 min. The samples were analysed by SDS-Pageand Western blotting.

Fractionation of bacterial cells

Fractionation of Y. enterocolitica cells was carried out asfollows. Bacterial cultures were induced for production ofYops in the presence of Ca2+. The cells were collected,washed, resuspended in saline buffer and disrupted by son-ication. The crude extract was centrifuged at 6000 r.p.m. for15 min to remove unbroken cells and aggregates. The super-natants were centrifuged again for 1 h at 100 000 g at 4∞C ina Centrikon T-1075 centrifuge (Kontron Instruments), and theproteins in supernatants and pellets were analysed by West-ern blot as described.

SycE-binding assay

The overlay assay to test binding of SycE in vitro was madeas described previously (Wattiau and Cornelis, 1993), usingcrude extracts of Top10 E. coli cells overexpressing SycE.This extract was obtained as follows. An aliquot from anovernight culture of Top10 E. coli cells transformed withpMAF33 was diluted in fresh LB medium and, after 1 h ofincubation at 37∞C, arabinose was added at a concentrationof 0.2%. The cells were incubated three additional hours andthen harvested and disrupted by sonication. The extract con-taining SycE was centrifuged and diluted 1:2 in PBS contain-ing 5% skim milk and applied to membrane-immobilizedYopE–DHFR fusion proteins. Binding of SycE was detectedwith rabbit polyclonal antiserum (Wattiau and Cornelis,1993).

Effect of aminopterin on secretion of fusion proteins

Yersinia enterocolitica DHOPEMT strain harbouring plasmidpMAF14 was plated on TSA plates supplemented with dif-ferent concentrations of apt and incubated for 24 h at 28∞Cin order to find the concentration of apt that inhibits the growthof the cells. Complete inhibition occurred at a concentrationof 1 mM Apt. To study the effect of apt on the secretion ofYopE52–DHFRwt, DHOPEMT Y. enterocolitica cells trans-formed with pMAF14 and R388 (Heidorn and Valentine,



1986) were precultured in BHI and diluted in BHI-Ox supple-mented with 1 mM apt. Bacteria were incubated for 4 h at37∞C, and the supernatants were analysed as describedabove.

Acknowledgements

We thank N. Grosdent for sharing the unpublished pNGB4,C. Youta for sequencing the constructs, and M. Folcher forhis collaboration in chromatography experiments. We arepleased to acknowledge Dr T. Langer (Munich) for plasmidpGEM4-DHFRmut and Dr G. Schatz (Basel) for the anti-DHFR antibody. We are grateful to J. Mota and N. Sauvonnetfor very useful discussions and suggestions, and to J.Ugalde, U. Jenal and E. Ceccarelli for critical reading of themanuscript. This work was supported by the Swiss NationalScience Foundation (grant 32-65393.01), the Belgian‘Fonds National de la Recherche Scientifique Médicale’(Convention 3.4595.97) and the Belgian ‘Direction généralede la Recherche Scientifique Communauté Française deBelgique’ (Action de Recherche Concertée 94/99-172). Thelaboratory also participates in the EU TMR network ‘MEMB-MACS’ (contract HRPN-CT-2000-00075). M.F.F. was therecipient of a ‘Societe Generale de Belgique’ ICP fellowship,and S.M. was a recipient of a Haas-Teichen ICP fellowship.

References

Anderson, D.M., and Schneewind, O. (1997) A mRNA signalfor the type III secretion of Yop proteins by Yersinia entero-colitica. Science 278: 1140–1143.

Birtalan, S., and Ghosh, P. (2001) Structure of the Yersiniatype III secretory system chaperone SycE. Nature StructBiol 8: 974–978.

Birtalan, S.C., Phillips, R.M., and Ghosh, P. (2002) Three-dimensional secretion signals in chaperone-effector com-plexes of bacterial pathogens. Mol Cell 9: 971–980.

Blocker, A., Jouihri, N., Larquet, E., Gounon, P., Ebel, F.,Parsot, C., et al. (2001) Structure and composition of theShigella flexneri ‘needle complex’, a part of its type IIIsecreton. Mol Microbiol 39: 652–663.

Boyd, A.P., Lambermont, I., and Cornelis, G.R. (2000a)Competition between the Yops of Yersinia enterocolitica fordelivery into eukaryotic cells: role of the SycE chaperonebinding domain of YopE. J Bacteriol 182: 4811–4821.

Boyd, A.P., Grosdent, N., Totemeyer, S., Geuijen, C., Bleves,S., Iriarte, M., et al. (2000b) Yersinia enterocolitica candeliver Yop proteins into a wide range of cell types: devel-opment of a delivery system for heterologous proteins. EurJ Cell Biol 79: 659–671.

Cheng, L.W., and Schneewind, O. (1999) Yersinia entero-colitica type III secretion. On the role of SycE in targetingYopE into HeLa cells. J Biol Chem 274: 22102–22108.

Clark, A.C., and Frieden, C. (1999) Native Escherichia coliand murine dihydrofolate reductases contain late-foldingnon-native structures. J Mol Biol 285: 1765–1776.

Cornelis, G.R., and Van Gijsegem, F. (2000) Assembly andfunction of type III secretory systems. Annu Rev Microbiol54: 735–774.

Cornelis, G., Vanootegem, J.C., and Sluiters, C. (1987)

Transcription of the yop regulon from Y. enterocoliticarequires trans acting pYV and chromosomal genes. MicrobPathog 2: 367–379.

Darwin, K.H., and Miller, V.L. (2001) Type III secretion chap-erone-dependent regulation: activation of virulence genesby SicA and InvF in Salmonella typhimurium. EMBO J 20:1850–1862.

Driessen, A.J. (2001) SecB, a molecular chaperone with twofaces. Trends Microbiol 9: 193–196.

Eilers, M., and Schatz, G. (1986) Binding of a specific ligandinhibits import of a purified precursor protein into mitochon-dria. Nature 322: 228–232.

Evdokimov, A.G., Tropea, J.E., Routzahn, K.M., and Waugh,D.S. (2002) Crystal structure of the Yersinia pestis GTPaseactivator YopE. Protein Sci 11: 401–408.

Frithz-Lindsten, E., Rosqvist, R., Johansson, L., andForsberg, A. (1995) The chaperone-like protein YerA ofYersinia pseudotuberculosis stabilizes YopE in the cyto-plasm but is dispensable for targeting to the secretion loci.Mol Microbiol 16: 635–647.

Hakansson, S., Schesser, K., Persson, C., Galyov, E.E.,Rosqvist, R., Homble, F., and Wolf-Watz, H. (1996) TheYopB protein of Yersinia pseudotuberculosis is essential forthe translocation of Yop effector proteins across the targetcell plasma membrane and displays a contact-dependentmembrane disrupting activity. EMBO J 15: 5812–5823.

Heidorn, J.V., and Valentine, C.R. (1986) Restriction enzymemap of cryptic plasmid accompanying pR711b and pR409;identity of pR409 and pR388. J Basic Microbiol 26: 621–625.

Hoiczyk, E., and Blobel, G. (2001) Polymerization of a singleprotein of the pathogen Yersinia enterocolitica into needlespunctures eukaryotic cells. Proc Natl Acad Sci USA 98:4669–4674.

Hynds, P.J., Robinson, D., and Robinson, C. (1998) The sec-independent twin-arginine translocation system can trans-port both tightly folded and malfolded proteins across thethylakoid membrane. J Biol Chem 273: 34868–34874.

Iriarte, M., and Cornelis, G.R. (1998) YopT, a new YersiniaYop effector protein, affects the cytoskeleton of host cells.Mol Microbiol 29: 915–929.

Kopytek, S.J., Dyer, J.C., Knapp, G.S., and Hu, J.C. (2000)Resistance to methotrexate due to AcrAB-dependentexport from Escherichia coli. Antimicrob AgentsChemother 44: 3210–3212.

Kovach, M.E., Elzer, P.H., Hill, D.S., Robertson, G.T., Farris,M.A., Roop, R.M., II, and Peterson, K.M. (1995) Fournew derivatives of the broad-host-range cloning vectorpBBR1MCS, carrying different antibiotic-resistance cas-settes. Gene 166: 175–176.

Kubori, T., Matsushima, Y., Nakamura, D., Uralil, J.,Lara-Tejero, M., Sukhan, A., et al. (1998) Supramolecularstructure of the Salmonella typhimurium type III proteinsecretion system. Science 280: 602–605.

Lee, V.T., and Schneewind, O. (2002) Yop fusions to tightlyfolded protein domains and their effects on Yersinia entero-colitica type III secretion. J Bacteriol 184: 3740–3745.

Luo, Y., Bertero, M.G., Frey, E.A., Pfuetzner, R.A., Wenk,M.R., Creagh, L., et al. (2001) Structural and biochemicalcharacterization of the type III secretion chaperones CesTand SigE. Nature Struct Biol 29: 29.



Menard, R., Sansonetti, P., Parsot, C., and Vasselon, T.(1994) Extracellular association and cytoplasmic partition-ing of the IpaB and IpaC invasins of S. flexneri. Cell 79:515–525.

Michiels, T., and Cornelis, G.R. (1991) Secretion of hybridproteins by the Yersinia Yop export system. J Bacteriol 173:1677–1685.

Narayana, N., Matthews, D.A., Howell, E.E., andNguyen-huu, X. (1995) A plasmid-encoded dihydrofolatereductase from trimethoprim-resistant bacteria has anovel D2-symmetric active site. Nature Struct Biol 2: 1018–1025.

Neyt, C., and Cornelis, G.R. (1999) Insertion of a Yop trans-location pore into the macrophage plasma membrane byYersinia enterocolitica: requirement for translocators YopBand YopD, but not LcrG. Mol Microbiol 33: 971–981.

Oefner, C., D'Arcy, A., and Winkler, F.K. (1988) Crystal struc-ture of human dihydrofolate reductase complexed withfolate. Eur J Biochem 174: 377–385.

Page, A.L., Sansonetti, P., and Parsot, C. (2002) Spa15 ofShigella flexneri, a third type of chaperone in the type IIIsecretion pathway. Mol Microbiol 43: 1533–1542.

Pfanner, N., and Geissler, A. (2001) Versatility of the mito-chondrial protein import machinery. Nature Rev Mol CellBiol 2: 339–349.

Pugsley, A.P. (1992) Translocation of a folded protein acrossthe outer membrane in Escherichia coli. Proc Natl AcadSci USA 89: 12058–12062.

Salvador, N., Aguado, C., Horst, M., and Knecht, E. (2000)Import of a cytosolic protein into lysosomes by chaperone-mediated autophagy depends on its folding state. J BiolChem 275: 27447–27456.

Sasso, S.P., Gilli, R.M., Sari, J.C., Rimet, O.S., and Briand,C.M. (1994) Thermodynamic study of dihydrofolate reduc-tase inhibitor selectivity. Biochim Biophys Acta 1207: 74–79.

Schesser, K., Frithz-Lindsten, E., and Wolf-Watz, H. (1996)Delineation and mutational analysis of the Yersiniapseudotuberculosis YopE domains which mediate translo-cation across bacterial and eukaryotic cellular membranes.J Bacteriol 178: 7227–7233.

Schlenstedt, G., Gudmundsson, G.H., Boman, H.G., andZimmermann, R. (1990) A large presecretory protein trans-locates both cotranslationally, using signal recognition par-ticle and ribosome, and post-translationally, without theseribonucleoparticles, when synthesized in the presence ofmammalian microsomes. J Biol Chem 265: 13960–13968.

Sory, M.P., and Cornelis, G.R. (1994) Translocation of ahybrid YopE-adenylate cyclase from Yersinia enterocoliticainto HeLa cells. Mol Microbiol 14: 583–594.

Sory, M.P., Boland, A., Lambermont, I., and Cornelis, G.R.

(1995) Identification of the YopE and YopH domainsrequired for secretion and internalization into the cytosol ofmacrophages, using the cyaA gene fusion approach. ProcNatl Acad Sci USA 92: 11998–12002.

Stebbins, C.E., and Galan, J.E. (2001) Maintenance of anunfolded polypeptide by a cognate chaperone in bacterialtype III secretion. Nature 414: 77–81.

Stueber, D., Ibrahimi, I., Cutler, D., Dobberstein, B., andBujard, H. (1984) A novel in vitro transcription-translationsystem: accurate and efficient synthesis of single proteinsfrom cloned DNA sequences. EMBO J 3: 3143–3148.

Teichmann, U., van Dyck, L., Guiard, B., Fischer, H.,Glockshuber, R., Neupert, W., and Langer, T. (1996) Sub-stitution of PIM1 protease in mitochondria by Escherichiacoli Lon protease. J Biol Chem 271: 10137–10142.

Tucker, S.C., and Galan, J.E. (2000) Complex function forSicA, a Salmonella enterica serovar typhimurium type IIIsecretion-associated chaperone. J Bacteriol 182: 2262–2268.

Vestweber, D., and Schatz, G. (1988) Point mutations desta-bilizing a precursor protein enhance its post- translationalimport into mitochondria. EMBO J 7: 1147–1151.

Walton, P.A., Hill, P.E., and Subramani, S. (1995) Import ofstably folded proteins into peroxisomes. Mol Biol Cell 6:675–683.

Wattiau, P., and Cornelis, G.R. (1993) SycE, a chaperone-like protein of Yersinia enterocolitica involved in Ohe secre-tion of YopE. Mol Microbiol 8: 123–131.

Wattiau, P., Bernier, B., Deslee, P., Michiels, T., andCornelis, G.R. (1994) Individual chaperones required forYop secretion by Yersinia. Proc Natl Acad Sci USA 91:10493–10497.

Wattiau, P., Woestyn, S., and Cornelis, G.R. (1996) Custom-ized secretion chaperones in pathogenic bacteria. MolMicrobiol 20: 255–262.

Wienhues, U., Becker, K., Schleyer, M., Guiard, B.,Tropschug, M., Horwich, A.L., et al. (1991) Protein foldingcauses an arrest of preprotein translocation into mitochon-dria in vivo. J Cell Biol 115: 1601–1609.

Woestyn, S., Allaoui, A., Wattiau, P., and Cornelis, G.R.(1994) YscN, the putative energizer of the Yersinia Yopsecretion machinery. J Bacteriol 176: 1561–1569.

Woestyn, S., Sory, M.P., Boland, A., Lequenne, O., andCornelis, G.R. (1996) The cytosolic SycE and SycH chap-erones of Yersinia protect the region of YopE and YopHinvolved in translocation across eukaryotic cell mem-branes. Mol Microbiol 20: 1261–1271.

Wulff-Strobel, C.R., Williams, A.W., and Straley, S.C. (2002)LcrQ and SycH function together at the Ysc type III secre-tion system in Yersinia pestis to impose a hierarchy ofsecretion. Mol Microbiol 43: 411–423.

SycE allows secretion of YopE-DHFR hybrids by the Yersinia enterocolitica type III Ysc system

Documents

Transcript of SycE allows secretion of YopE-DHFR hybrids by the Yersinia enterocolitica type III Ysc system