Tài liệu Báo cáo Y học: Identification and characterization of the Escherichia coli stress protein

Identification and characterization of the Escherichia coli stress

protein UP12, a putative in vivo substrate of GroEL

Elena S. Bochkareva, Alexander S. Girshovich and Eitan Bibi

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Many groups of proteins play important roles in the cell’s

response to various stresses. The molecular chaperone

GroEL of Escherichia coli represents one such highly con￾served family of stress proteins. We have observed that iso￾lated GroEL complexes from stationary cultures contain

various polypeptides that can be released from the chap￾eronin by GroES and/or ATP, and identified two such

polypeptides as the proteins GatY and UP12. Whereas

GatY had been isolated previously, as an in vivo substrate of

GroEL, the isolation of UP12 in a complex with GroEL was

intriguing, because based on sequence similarity it was sug￾gested that UP12 might also be a functional stress protein.

UP12 belongs to a family of universal stress proteins

(UspA family), of which UspA itself, and three additional

paralogues, have been characterized previously. Here we

show that UP12 accumulates under various growth inhibi￾tory conditions and induced by heat shock. Furthermore,

unlike wild-type cells, a UP12 deletion mutant recovers

slowly from late stationary growth conditions, and has a

marked sensitivity to the toxic agent carbonyl cyanide

m-chlorophenyl hydrazone (CCCP). Finally, coimmuno￾precipitation experiments confirmed the initial observation

that UP12 interacts with GroEL. Therefore, we suggest that

UP12 may function as a universal stress protein, interaction

of which with GroEL possibly ensures its proper folding

state.

Keywords: GroEL substrate; UP12; universal stress protein;

Stress response; E.coli.

Escherichia coli cells undergo a transition from a rapid

growth phase to a stationary phase, which is accompanied

by a variety of physiological changes that affect gene

expression, the structure and composition of the cell wall,

DNA organization, synthesis of storage compounds such as

glycogen and polyphosphate, and other cellular processes

[1,2]. As a result of these changes, the cells become resistant

to various deleterious stresses such as heat shock, UV

irradiation, acidic or basic conditions, osmotic shock, and

oxidation [3–5].

Studies carried out in several laboratories have identified

specific cellular networks and individual genes expressed in

the stationary growth phase that improve the survival of

E.coli during prolonged periods of starvation and other

stress conditions [6–11]. One of these genes is uspA, which

encodes a small cytoplasmic protein, UspA (universal stress

protein A) that is unique in its universal responsiveness to

diverse stresses [12]. The synthesis of UspA is greatly

increased under growth inhibitory conditions, including the

depletion of essential nutrients or exposure to various toxic

agents. Moreover, E.coli carrying an inactivated uspA is

more sensitive to prolonged growth inhibition caused by a

variety of starvation and other stress conditions [13,14].

In the course of systematically analyzing the sequenced

E.coli genome [15], it has been found that five ORFs share

some homologies with UspA. Two of them, encoded by

ybdQ and ynaF, were previously identified as unknown

proteins (UP12 and UP03, respectively) by 2D-PAGE [16].

Three E.coli paralogues of UspA have been characterized

recently [17], and the results of this study showed that UspA

is a prototype for a family of conserved proteins (universal

stress proteins) found not only in bacteria but also in other

organisms.

Other groups of proteins also play important roles in

bacterial stress response. One important group includes the

heat-shock proteins, whose induction under stress conditions

in E.coli requires the heat-shock transcription factor r32

(rpoH gene product) [18]. Many heat-shock proteins, such as

members of the Hsp70 and Hsp60 protein families, are

molecular chaperones. Functionally, they bind to non-native

structural forms of various polypeptides and assist them in

reaching a native conformation [19]. Consequently, as

molecular chaperones, they prevent misfolding and aggre￾gation of unfolded proteins under heat-shock and other

stress conditions [20,21]. The E.coli heat-shock protein

GroEL belongs to the highly conserved Hsp60 family of

oligomeric molecular chaperones named chaperonins [22].

GroEL andits small cohortGroES were found to be essential

not only under stress, but also for growth under all

experimental conditions tested to date [23]. GroEL transi￾entlyinteracts (in aGroES- andMgATP-dependentmanner)

with many unfolded newly synthesized proteins in vitro and

in vivo [24–26]. Among the proposed physiological substrates

of GroEL are structurally unstable proteins that require

GroEL for permanent conformational maintenance [27].

In the course of GroEL purification from stationary

cultures of E.coli, we noticed that a few polypeptides

Correspondence to E. Bochkareva, Department of Biological

Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Fax: + 972 89 344118, Tel.: + 972 89 342912,

E-mail: [email protected]

Abbreviations: CCCP, carbonyl cyanide m-chlorophenyl hydrazone;

DNP, a-dinitrophenol; DM, n-dodecyl-b,D-maltoside; IPTG, isopro￾pyl b-D-thiogalactopyranoside.

(Received 15 February 2002, revised 25 April 2002,

accepted 3 May 2002)

Eur.J.Biochem. 269, 3032–3040 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.02978.x