AbstractE. coli trigger factor is an abundant cytosolic protein originally iDAntified by its ability to maintain the precursor of a secretory protein in a translocation competent form. Recent studies shed new light on the function of this protein. Trigger factor was found to be a peptidyl-prolyl-cisltrans-isomerase capable of catalysing protein folding in vitro, to associate with nascent cytosolic and secretory polypeptiDA chains, and to cooperate with the GroEL chaperone in promoting proteolysis of an unstable polypeptiDA in vivo. These findings suggest roles for trigger factor in various folding processes of secretory as well as cytosolic proteins

Bukau, Bernd

Hesterkamp, Thomas

Elsevier - Publisher Connector 

The Escherichia coli trigger factor 

FEBS Letters 389 (1996) 32-34 FEBS 17191 
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The Escherichia coli trigger factor 
Thomas Hesterkamp, Bernd Bukau* 
Zentrum ffir Molekulare Biologie der Universitiit Heidelberg, D-69120 Heidelberg, Germany 
Received 13 May 1996 
Abstract E. coli trigger factor is an abundant cytosolic protein 
originally identified by its ability to maintain the precursor of a 
secretory protein in a translocation competent form. Recent 
studies shed new light on the function of this protein. Trigger 
factor was found to be a peptidyl-prolyl-cisltrans-isomerase 
capable of catalysing protein folding in vitro, to associate with 
nascent cytosolic and secretory polypeptide chains, and to 
cooperate with the GroEL chaperone in promoting proteolysis 
of an unstable polypeptide in vivo. These findings suggest roles 
for trigger factor in various folding processes of secretory as well 
as cytosolic proteins. 
Key words: Ribosome; Prolyl isomerase; Chaperone; Protein 
folding; Translocation; Proteolysis 
1. Discovery 
Trigger factor was discovered in a biochemical screen for 
cytosolic components of the secretion machinery of E. coli. 
Analysing the translocation of the outer membrane protein A 
(OmpA) across inner membrane vesicles in vitro, Wickner and 
coworkers identified an activity that stabilized the precursor 
(proOmpA) in a loosely folded conformation thereby stimu- 
lating its membrane translocation. This activity was shown to 
reside in a monomeric protein with an apparent molecular 
weight of 60 kDa, termed trigger factor [1 3]. Trigger factor 
formed stable 1:1 complexes with chemically denatured 
proOmpA that was diluted from denaturant. When proOmpA 
was allowed to fold in the absence of trigger factor, however, 
translocation of proOmpA could not be restored, indicating 
that trigger factor could not actively unfold the substrate [2]. 
2. Genetic analysis 
The tig gene encoding trigger factor was cloned [4] and its 
expression found to be growth phase controlled and thus co- 
regulated with genes encoding ribosomal components (F. 
Neidhardt, personal communication cited in [4]). Genetic ana- 
lysis of the in vivo function of trigger factor using a condi- 
tional depletion strain failed to provide new insights. At con- 
ditions depleting trigger factor to less than 5% of the normal 
levels, cells remain fully viable but form filaments indicative of 
cell division defects. No defects in the translocation of 
proOmpA, even in a trigger factor depletion strain with an 
additional deficiency for the secretion specific SecB chaperone, 
were observed [4]. These results suggest hat trigger factor is 
dispensable for growth of E. coli, although analysis of a rig 
*Corresponding author. Fax: (49) (6221) 545892. 
E-mail: bukau@sun0.urz.uni-heidelberg.de 
knockout mutant is required for a proof. It is not excluded 
either that trigger factor does play an important role in me- 
tabolism but that backup systems exist replacing missing trig- 
ger factor functions under depletion conditions. An important 
physiological role of trigger factor is indicated by the finding 
that tig homologs exist in other bacterial genomes [5-7] in- 
cluding that of Mycoplasma genitalium which is believed to 
contain the minimum set of genes required for life [7]. 
3. Trigger factor associates with nascent polypeptide chains and 
has prolyl isomerase activity 
Work in three laboratories, aimed at investigating different 
processes related to protein folding in vivo, converged in the 
discovery of novel features of trigger factor that changed our 
view of its biological function. A search by Fischer and co- 
workers for a ribosome-bound peptidyl-prolyl-cis/trans-iso- 
merase (PPIase) activity led to the identification of trigger 
factor [8]. In an in vitro refolding assay using a RNaseT1 
mutant protein as substrate trigger factor accelerated the 
rate-limiting prolyl isomerisation step more efficiently than 
any other known PPIase. Using oligopeptide substrates the 
specificity of trigger factor was determined to resemble that 
of immunophilins of the FK506 binding protein (FKBP) fa- 
mily. However, the failure of the FK506 immunosuppressant 
to inhibit the PPIase activity of trigger factor led these 
authors to propose that trigger factor constitutes a novel fa- 
mily of PPIases [8]. 
Luirink and coworkers used an in vitro translation system 
to investigate the interactions of the E. coli signal recognition 
particle (SRP) with a variety of nascent secretory polypeptide 
chains by crosslinking [9]. In addition to crosslinks of nascent 
polypeptide chains with the P48 protein component of SRP, 
they observed prominent crosslinked products originating 
from interaction of trigger factor with nascent chains. P48 
and trigger factor competed for crosslinking to prePhoE sug- 
gesting overlapping substrate specificities of both proteins. In 
contrast o P48, however, trigger factor was also efficiently 
crosslinked to non-secretory nascent chains [9]. 
Searching for E. coli proteins associated with nascent poly- 
peptide chains of 13-galactosidase in an in vitro transcription- 
translation system, Bukau and coworkers observed a salt-re- 
sistant association of trigger factor with translating ribosomes 
[10]. This association was sensitive to puromycin treatment 
and thus dependent on the presence of nascent 13-galactosi- 
dase. A physical association of trigger factor with nascent 
chains of preprolactin and a non-secretory preprolactin mu- 
tant was demonstrated by crosslinking. For the non-secretory 
substrate, trigger factor was the single major crosslinking 
component of the E. coli cytosol. These authors noted a 
homology of trigger factor with PPIases of the FKBP family 
0014-5793/96/$12.00 © 1996 Federation of European Biochemical Societies. All rights reserved. 
PII S00 1 4 -5793(96)00582-0  
T. Hesterkamp, B. Bukau/FEBS Letters 389 (1996) 32-34 33 
and demonstrated PPIase activity for purified trigger factor. 
Together, a role for trigger factor as a co-translationally act- 
ing folding catalyst was proposed [10]. 
4. Trigger factor binds to ribosomes 
The ability of trigger factor to associate with nascent poly- 
peptide chains raises the question of the mechanism of its 
targeting to the substrate. Crosslinking of trigger factor with 
nascent chains was abolished after their puromycin-mediated 
release from the ribosome [9,10]. Thus, either misfolding of 
the puromycyl fragments or loss of the ribosomal environ- 
ment prevented substrate association with trigger factor under 
these conditions. Trigger factor has indeed been shown to 
bind to the isolated large ribosomal subunit [11]. This subunit 
contains the exit site for nascent polypeptide chains and it is 
tempting to speculate that trigger factor binding to the ribo- 
some in the vicinity of the nascent chain exit site allows its 
efficient association with the substrate. 
It is important to note that the association of trigger factor 
with non-translating ribosomes is salt-sensitive, in contrast o 
its salt-resistant association with translating ribosomes in vitro 
[10]. There are different possible explanations for this finding. 
First, it is possible that the association of trigger factor with 
nascent chains stabilizes the trigger factor ribosome complex. 
The PPIase activity, however, is unlikely to contribute to this 
stabilisation since PPIases generally [12] and the PPIase do- 
main of trigger factor (T.H. and B.B., unpublished) do not 
form stable complexes with their substrates. Alternatively, the 
substantial conformational changes in ribosomes leading to 
their tightly coupled state during translation might increase 
the affinity for trigger factor. In this respect the observed 
puromycin-mediated r lease of trigger factor from translating 
ribosomes [10] may result from conformational changes un- 
coupling the ribosome. Consistent with this interpretation is 
our observation that trigger factor disappeared at physiologi- 
cal salt concentrations from ribosomes, isolated from E. coli 
cultures as run-off ribosomes, when the ribosomal subunits 
were uncoupled by magnesium chelation. In contrast, in the 
presence of magnesium this complex was resistant o treat- 
ment with 1 M potassium chloride (T.H. and B.B., unpub- 
lished). Taken together, it is conceivable that the coupling 
status of ribosomes during the translation process influences 
the binding of trigger factor. 
5. Structure-function relationship of trigger factor's PPIase 
activity 
Hesterkamp et al. and Callebaut and Mornon reported on a 
homology of trigger factor with PPIases of the FKBP family 
[10,13]. The region of homology is located within the central 
part of the trigger factor polypeptide chain, between residues 
165 and 240, and is restricted to residues forming the sub- 
strate binding pocket in human FKBP12 [14-16]. The PPIase 
domain of trigger factor exhibits a higher degree of conserva- 
tion in Escherichia coli, Campylobacter jejuni, Haemophilus 
influenzae and Mycoplasma genitalium than full-length trigger 
factor, indicating the functional importance of this domain 
[171. 
These homology predictions were recently confirmed by 
functional analyses of trigger factor fragments obtained by 
limited proteolysis with endoproteinase Glu-C (V8) [17] and 
subtilisin [18]. Proteolytic fragments of 12.8 kDa (Va1132 to 
Glu247) and 11.8 kDa (Arga45 to Glu 251), respectively, dis- 
played a specific PPIase activity similar to that of full-length 
trigger factor. An even smaller recombinant fragment com- 
prising only 102 amino acids (Glu148 to Thr249) also displayed 
full PPIase activity [18]. The size of this PPIase domain is in 
good agreement with the size of the prototype FKBP12. A 
number of non-conservative amino acid exchanges between the 
trigger factor FKBP-domain and FKBP12 may account for 
the observed resistance of the PPIase activity of trigger factor 
to the immunosuppressive drugs FK506 and rapamycin [8]. 
Assuming a modular structure of trigger factor, the central 
PPIase domain would be flanked by two almost equally sized 
N- and C-terminal domains. While a proteolytic fragment of 
14 kDa apparent molecular weight starting with the authentic 
N-terminus of trigger factor has been reported [18], the poly- 
peptide chain C-terminal to the PPIase domain is highly sus- 
ceptible to proteolysis [17,18]. The functional roles of these 
domains remain to be elucidated. 
6. Trigger factor interacts with GroEL in the degradation of 
abnormal proteins 
A different function for trigger factor has been proposed by 
work from the Goldberg laboratory aimed at understanding 
the role of chaperones in protein degradation. The fusion 
protein CRAG, composed of protein segments of ~, cro, pro- 
tein A and [~-galactosidase, is degraded in vivo by the ClpP 
protease in a process stimulated by the GroEL chaperonin 
[19]. Using conditional trigger factor depletion or overproduc- 
tion strains it has now been shown that trigger factor also 
accelerates the degradation of CRAG. Furthermore, a physi- 
cal interaction of GroEL and trigger factor with this substrate 
has been demonstrated on a CRAG affinity column [20]. Ad- 
dition of ATP to elute GroEL from the affinity column re- 
sulted in co-elution of trigger factor and both proteins re- 
mained associated in a subsequent gel filtration step. This 
finding was interpreted to indicate that pre-existing GroEL- 
trigger factor complexes bound to CRAG [20]. Even in the 
absence of the artificial substrate, GroEL and trigger factor 
co-purified from E. coli lysates [20]. These observations raise 
the possibility that trigger factor plays a role in targeting 
substrates to the GroEL chaperonin. This might be required 
for the efficient folding to the native state of a subset of newly 
synthesized proteins as well as for the assisted folding or de- 
gradation of misfolded proteins. 
7. A chaperone function of trigger factor? 
The original observation that trigger factor forms stable 1 : 1 
complexes with unfolded proOmpA is reminiscent of a cha- 
perone-like activity. This is further supported by the finding 
that the activity of trigger factor in preventing misfolding of 
the substrate in vitro can be replaced by the activities of the 
SecB and GroEL chaperones [21]. It is also remarkable in this 
context that trigger factor outscores by far all other known 
PPIases in the efficiency of catalysis of the isomerisation of 
the single cis-proline residue during RNaseT1 refolding [8]. 
Trigger factor had lower activity in catalysing the isomerisa- 
tion of a cis-proline residue in a tetrapeptide of similar local 
sequence [8]. It is thus conceivable that trigger factor recog- 
nizes and modulates the polypeptide chain surrounding the 
34 
critical proline residue, eventually as part of a chaperone ac- 
tivity, to increase the efficiency of prolyl isomerisation cataly- 
sis. This hypothesis is further supported by the intriguing 
finding that trigger factor-mediated refolding of RNaseT1 
can be competitively inhibited by addition of the chaperone 
substrate reduced carboxymethylated lactalbumin (F.X. 
Schmid, personal communication). Differences, however, exist 
between trigger factor and the well known DnaK and GroEL 
chaperone systems in that trigger factor fails to prevent the 
aggregation of refolding firefly luciferase (T.H. and B.B., un- 
published). 
Although quite speculative at this point, the idea of trigger 
factor being a ribosome-associated folding factor which accel- 
erates prolyl isomerisation and stabilizes the folding chain 
until it is completed or handed over to other chaperones i  
highly attractive and will trigger future experiments. 
Acknowledgements: Work in the laboratory was supported by grants 
from the Forschungsschwerpunkt des Landes Baden-Wtirttemberg, 
the DFG, and the Fonds der Chemischen I dustrie to B.B. and a 
fellowship from the Boehringer Ingelheim Fonds to T.H. 
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The Escherichia coli trigger factor

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