Báo cáo khoa học: The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum ) a quantitative approach potx

The heat shock protein 70 molecular chaperone network
in the pancreatic endoplasmic reticulum
)
a quantitative
approach
Andreas Weitzmann, Christiane Baldes, Johanna Dudek and Richard Zimmermann
Medizinische Biochemie und Molekularbiologie, Universita
¨
t des Saarlandes, Homburg, Germany
The initial step in the biogenesis of approximately
30% of eukaryotic proteins is their integration into
the membrane or their transport into the lumen of the
endoplasmic reticulum (ER). Protein integration or
transport into the ER can occur cotranslationally or
post-translationally, and typically requires signal pep-
tides at the N-terminus of the precursor proteins and
the transport machinery. Post-translational protein
transport into the yeast ER involves the Sec complex
in the membrane, comprising the Sec61p subcomplex
[1,2], the putative signal peptide receptor subcomplex
[3,4], the heat shock protein (Hsp) 40, termed Sec63p
[5], and the luminal Hsp70 proteins Kar2p [6] and
Lhs1p [7]. Sec63p and Kar2p are essential proteins in
yeast and, together with Lhs1p, are also involved in
cotranslational protein transport into the ER [8,9].
Cotranslational protein transport into dog pancreas
microsomes involves a similar Sec61 complex [10–12].
Furthermore, protein transport into the mammalian
ER involves Hsp70-type molecular chaperones and
their Hsp40-type cochaperones: Hsp70-type molecular
chaperones of the ER lumen, IgG heavy chain-binding

protein [BiP (also termed glucose-regulated protein
(Grp) 78, HspA5) and Grp170 (Orp150) are involved
in cotranslational and post-translational insertion of
precursor polypeptides into the Sec61 complex [13].
Keywords
BiP; endoplasmic reticulum; J-domains;
nucleotide exchange factors; molecular
chaperones
Correspondence
R. Zimmermann, Medizinische Biochemie
und Molekularbiologie, Universita
¨
t des
Saarlandes, D-66421 Homburg, Germany
Fax: +49 6841 1626288
Tel: +49 6841 1626510
E-mail:
(Received 15 June 2007, revised 9 August
2007, accepted 10 August 2007)
doi:10.1111/j.1742-4658.2007.06039.x
Traditionally, the canine pancreatic endoplasmic reticulum (ER) has been
the workhorse for cell-free studies on protein transport into the mamma-
lian ER. These studies have revealed multiple roles for the major ER-lumi-
nal heat shock protein (Hsp) 70, IgG heavy chain-binding protein (BiP), at
least one of which also involves the second ER-luminal Hsp70, glucose-reg-
ulated protein (Grp) 170. In addition, at least one of these BiP activities
depends on Hsp40. Up to now, five Hsp40s and two nucleotide exchange
factors, Sil1 and Grp170, have been identified in the ER of different mam-
malian cell types. Here we quantified the various proteins of this chaperone
network in canine pancreatic rough microsomes. We also characterized the

various purified proteins with respect to their affinities for BiP and their
effect on the ATPase activity of BiP. The results identify Grp170 as the
major nucleotide exchange factor for BiP, and the resident ER-membrane
proteins ER-resident J-domain protein 1 plus ER-resident J-domain pro-
tein 2 ⁄ Sec63 as prime candidates for cochaperones of BiP in protein trans-
port in the pancreatic ER. Thus, these data represent a comprehensive
analysis of the BiP chaperone network that was recently linked to two
human inherited diseases, polycystic liver disease and Marinesco–Sjo
¨
gren
syndrome.
Abbreviations
BiP, IgG heavy chain-binding protein; ER, endoplasmic reticulum; ERj, endoplasmic reticulum-resident J-domain protein; Grp, glucose-
regulated protein; GSH, glutathione; GST, glutathione S-transferase; Hsp, heat shock protein; RM, rough microsome; SPR, surface plasmon
resonance.
FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5175
BiP was also identified as a luminal protein that is
involved in the completion of protein translocation
[14,15]. Furthermore, BiP was shown to seal the lumi-
nal end of the mammalian Sec61 complex in the
absence of protein translocation and at several stages
during cotranslational translocation of preproteins
[16–18]. BiP was shown to involve an unidentified resi-
dent ER Hsp40 and could not be substituted by its
yeast ortholog Kar2p in Sec61 gating [18]. A mamma-
lian ortholog of yeast protein Sec63p was shown to be
an abundant protein in canine pancreatic microsomes
and was found in association with the Sec61 com-
plex [19–21]. The Sec63-related protein ER-resident
J-domain protein (ERj) 1 was observed to be asso-

ciated with translating ribosomes on the ER surface
[22–24] and to be able to complement a yeast mutant
that is deficient in Sec63p [25].
In the ER of Saccharomyces cerevisiae, four Hsp40
proteins with a luminal J-domain have been identified:
the two membrane proteins Sec63p [5] and Scj2p [26],
and the two luminal proteins Scj1p [27] and Jem1p
[28]. In the ER of various mammalian cells, five Hsp40
proteins have been identified: the three membrane pro-
teins Sec63 (alternative names: ERj2, DnaJC2) [19–21],
ERj1 (Mtj1p, DnaJC1) [22,29,30], and ERj4 (MDG1,
DnaJB9) [31,32], and the two luminal proteins ERj3
(HEDJ, Dj9, DnaJB11) [33–35] and ERj5 (JPDI,
DnaJC10) [36,37] (Fig. 1, Table 1). Furthermore,
nucleotide exchange factors ) Sil1p in yeast [38,39]
and Sil1 (also termed BAP) in mammalian cells
[40] ) have been identified, and Lhs1p and Grp170
were shown to be alternative nucleotide exchange
factors for Kar2p and BiP, respectively [41,42]. Both
LHS1 and SIL1 are nonessential genes in yeast. How-
ever, simultaneous deletion of both genes results in
synthetic lethality.
Here we quantitatively characterized the Hsp70
chaperone network in the rough ER of a single
J domain
N
C
N
C
cytosol

ER lumen
J-domainJ-domain
N
C
J domain
J domain
ERj2/Sec63
ERj1
ERj4
ERj3 ERj5
N
C
4
TRX
Cys
GF
GF
N
C
Grp170
BiP
Sil1
N
N
N
C
C
C
ER membrane
ATPase

domain
ATPase
domain
PB
domain
PB
domain
Fig. 1. The established network of Hsp70-type molecular chaper-
ones in the lumen of the mammalian ER. The cartoon summarizes
data from different cell types. The putative domain organization of
the various proteins is indicated (PB, peptide-binding domain; GF,
glycine ⁄ phenylalanine-rich region; Cys, cysteine-rich region; TRX,
thioredoxin-like domains). The quantitative aspects are summarized
in Table 1.
Table 1. Hsp70 chaperones and their cochaperones of the ER in the canine pancreas. The concentrations refer to a suspension of RMs,
with a concentration of heterotrimeric Sec61 complexes of 2.12 l
M [54]. The affinities of Hsp40s for BiP are based on the SPR experiments
shown in Fig. 4 or were determined previously [21,22]. The ATPase experiments shown in Fig. 5 are the basis for the stimulatory effects of
Hsp40, Sil1, and Grp170. The experimental details are given in Experimental procedures. ND, not determined.
Protein
(alternative name)
Concentration
in suspension
of RMs (l
M)
Recombinant protein
(amino acid residues)
Affinity for BiP
in the presence
of ATP

(K
D
in lM)
Stimulation of
ATPase activity
of BiP
(fold)
Further stimula-
tion of BiP
ATPase by
Grp170
(fold)
Sil1
(fold)
BiP (Grp78, HspA5) 5.00 BiP)6His (20–655) – – 1 1.5
Grp170 (Orp150) 0.60 – ND 1 – –
ERj1 (Mtj1p, DnaJC1) 0.36
ERj1J GST–J-domain (44–140) 0.12 5.2 5.6 2.4
ERj2 (Sec63p, DnaJC2) 1.98
ERj2J GST–J-domain (91–189) 5.00 3.5 5.7 2.2
ERj3 (HEDJ, Dj9, DnaJB11) 0.29 GST–ERj3 (18–336) 3.60 1.8 1.9 1.3
ERj3J GST–J-domain (18–119) 3.50 ND ND ND
ERj4 (MDG1, DnaJB9) 0 GST–ERj4 (23–222) 6.07 1.6 1.5 1
ERj5 (JPDI, DnaJC10) 2.00 GST–ERj5 (26–793) 0.45 3.9 ND 1.1
ERj5J GST–J-domain (26–113) 0.59 1.8 4.3 ND
Sil1 (BAP) 0.005 GST–Sil1 (39–461) ND 1.5 – –
Pancreatic endoplasmic reticulum chaperone network A. Weitzmann et al.
5176 FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS
mammalian tissue, canine pancreas, which predomi-
nantly comprises exocrine cells. Except for ERj4, all

mammalian Hsp40s of the ER were detected. As Sil1
was found at very low concentrations, Grp170 (ortho-
log of yeast Lhs1p) appears to act as the predominant
nucleotide exchange factor for BiP–ADP in the pancre-
atic ER. The interactions of the various J-domains
with BiP were characterized by pull-down experiments,
surface plasmon resonance (SPR) spectroscopy, and
ATPase experiments. These data provide the first
comprehensive and quantitative analysis of a chaper-
one network that was recently linked to two human
hereditary diseases, autosomal dominant polycystic
liver disease (OMIM 174050) and the neurodegener-
ative Marinesco–Sjo
¨
gren syndrome (OMIM 248800)
[43–46].
Results
Two Hsp70s, four Hsp40s and Sil1 form a
chaperone network in the pancreatic ER
Previously, we had determined the concentrations of
BiP, Grp170, ERj1, ERj2 and ERj3 in suspensions of
dog pancreas microsomes [21,22,33,34]. In order to
determine how abundant ERj4, ERj5 and Sil1 are in
these pancreatic microsomes, purified glutathione
S-transferase (GST) hybrid proteins and specific anti-
bodies were employed in western blotting, according
to the established procedure (Fig. 2; Table 1). In all
cases, two different antibodies were used that recog-
nized the respective recombinant protein. In the case
of ERj5 and Sil1, these antibodies recognized an iden-

tical band in the pancreatic microsomes. We deter-
mined concentrations of 2 lm for ERj5 and 5 nm for
Sil1 in the microsomal suspensions. In the case of
ERj4, the antibodies failed to identify a common
antigen in the pancreatic microsomes. Therefore, we
conclude that ERj4 is not an abundant protein in
dog pancreas microsomes under physiological condi-
tions. This view is also supported by the fact that we
failed to characterize ERj4 in proteomic analysis of
these microsomes. It follows from the concentrations
of Hsp70 and Hsp40 proteins in the ER lumen
(Table 1) that all Hsp40s can be associated with BiP
at any given time.
The J-domains of all mammalian ER-resident
Hsp40s productively interact with BiP but differ
in their affinities for BiP
To determine whether the mammalian ER-resident
Hsp40s contain a functional J-domain, hybrid proteins
0
500
1000
1500
02468
0
100
200
300
400
A
B

00,511,522,5
microsomes (µL)
colour intensity (arbitrary units)
protein (µg)
colour intensity (arbitrary units)
Fig. 2. Quantitation of proteins in dog pancreas microsomes. Serial
dilutions of BSA (filled squares) were run on SDS polyacrylamide
gels in parallel with two samples of purified recombinant protein
(arrowheads, A). The proteins were stained with Coomassie Brilliant
Blue, and the staining intensity was quantified by densitometry (Per-
sonal Densitometer; Applied Biosystems, Krefeld, Germany). The
same purified protein (arrowhead) was run on SDS polyacrylamide
gels in parallel with serial dilutions of dog pancreas microsomes
(filled circles, B). Subsequently, the proteins were transferred to
poly(vinylidene difluoride) membranes and incubated with rabbit
antibodies that were directed against the protein of interest and
with a peroxidase conjugate of goat anti-(rabbit IgG) serum. The
bound antibodies were made visible by incubation with enhanced
chemiluminescence (ECL) and exposure to X-ray film. The intensity
of silver precipitation was quantified by densitometry. The calcula-
tion of the molar concentration of the respective protein in micro-
somal suspensions was based on the predicted molecular mass of
the protein, as calculated by the protean option of the
LASERGENE
DNASTAR
sequence analysis software (GATC, Konstanz, Germany).
A. Weitzmann et al. Pancreatic endoplasmic reticulum chaperone network
FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5177
ABC
D

G
EF
Pancreatic endoplasmic reticulum chaperone network A. Weitzmann et al.
5178 FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS
comprising GST and the respective protein or
J-domain were constructed, purified, and subjected to
three activity assays. In the case of the membrane pro-
teins ERj1, ERj 2, and ERj4, the transmembrane
domains were absent form the GST hybrids (Table 1).
Thus, only the ER-luminal domains were analyzed in
these cases. In the case of the two luminal Hsp40s
ERj3 and ERj5, GST hybrids were analyzed that con-
tained either the J-domains or the full-length proteins.
When compared to each other, the two types of GST
hybrids behaved quite similarly in the functional assays
that were employed here (Table 1).
In the first series of experiments, ‘pull-down assays’
were carried out with detergent extracts of dog pan-
creas microsomes as described previously [21]. GST
served as a negative control. GST or GST hybrids
were immobilized on glutathione (GSH)–Sepharose
and incubated with detergent extracts of dog pancreas
microsomes in the absence or presence of ATP. The
bound proteins were eluted and subjected to
SDS ⁄ PAGE and subsequent staining with Coomassie
Brilliant Blue (Fig. 3). All GST hybrids selectively
pulled down BiP from the detergent-solubilized micro-
somal proteins in the presence of ATP and less
efficiently in its absence (Fig. 3A–F, lane 6 versus
lane 4). From our results, we conclude that BiP inter-

acts with all ERjs in a productive manner, as: (a) GST
did not pull down BiP (Fig. 3G, lanes 4 and 6); and
(b) the other major molecular chaperones, present in
the detergent extract of dog pancreas microsomes
(such as Grp94 and calreticulin), did not bind to the
GST hybrids (Fig 3A–F, lane 6 versus lane 5). We
note, however, that the different GST hybrids were dif-
ferent in their BiP pull-down efficiencies (see below).
We next characterized the interaction of BiP with
the GST hybrids by SPR spectroscopy as described
previously [21] (Fig. 4). We determined the apparent
affinities in the presence of ATP (K
D
), which are given
in Table 1. In summary, BiP has an approximately 10-
fold higher affinity for ERj1 and ERj5 as compared to
ERj2, ERj3 and ERj4. However, we note that these
apparent affinities have to be treated with caution, as
the kinetics could not be fitted perfectly to a 1 : 1
binding model. The generally accepted explanation for
this fact is that after J-domain-mediated ATP hydroly-
sis and in the absence of a real substrate, BiP binds
Hsp40 as a substrate [47]. Accordingly, this interaction
is not seen when only the ATPase domain of BiP is
employed instead of full-length BiP (data not shown).
We note that it was also observed for yeast Kar2p plus
Sec63p that a stable interaction between this Hsp70–
Hsp40 pair is possible in the absence of any substrate
polypeptide [47], and stable interactions were also seen
previously between BiP and mammalian ERj2 ⁄ Sec63

[21] and ERj1 [22], in both pull-down and SPR experi-
ments.
Next, we investigated whether the GST hybrids stim-
ulate BiP’s ATPase activity under steady-state condi-
tions, i.e. in the presence of 500 lm ATP. BiP was
incubated with [
32
P]ATP[cP] in the absence or presence
of GST hybrid. After various times of incubation, the
samples were analyzed by TLC and phosphorimaging
(Fig. 5A–E; Table 1). According to the time-dependent
hydrolysis of ATP under the different conditions, all
J-domains stimulated the ATPase activity of BiP. GST
had no such stimulatory effect, even at much higher
concentrations [21]. Therefore, it seems to be unlikely
that the observed stimulation of BiP’s ATPase activity
by the GST–J-domain hybrid was due to a BiP–sub-
strate rather than a BiP–cochaperone interaction. In
the case of ERj1, ERj3, and ERj4, the stimulatory
effects of the ERjs correlated with their affinities for
BiP. However, for unknown reasons, this was not the
case for ERj2 ⁄ Sec63 and ERj5.
Grp170 serves as an efficient and general
nucleotide exchange factor for BiP
Our previous attempts to purify Grp170 with ATP-
affinity chromatography resulted in a mixture of
Grp170 and BiP [13]. Here, we employed gel filtration
chromatography in the absence or presence of ATP as
a subsequent and final purification step (Fig. 6A,B). In
the absence of ATP, a proportion of BiP cofractionat-

ed with Grp170, with an elution maximum of both
proteins at a position that corresponded to a molecular
mass of 240 kDa (Fig. 6A). In addition, the vast
majority of BiP was observed at a position that corre-
sponded to its monomeric molecular mass (70 kDa).
In the presence of ATP, however, there was hardly
any overlap of the two proteins, and they more or less
eluted according to their theoretical molecular masses
(70 and 140 kDa; Fig. 6B). Apparently, the two Hsp70-
type molecular chaperones can form a heterodimeric
Fig. 3. Functional characterization of Hsp40-type cochaperones of the ER: selective binding of BiP. GST or GST hybrid was immobilized and
incubated with detergent extract of microsomes in the absence or in the presence of ATP as described in Experimental procedures. The
unbound and bound proteins were collected and subjected to SDS ⁄ PAGE and subsequent staining with Coomassie Brilliant Blue. We note
that: (a) the band that is labeled BiP was identified as such by western blotting; and (b) we failed to detect Grp170 in the bound fractions
under these conditions.
A. Weitzmann et al. Pancreatic endoplasmic reticulum chaperone network
FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5179
Fig. 4. Functional characterization of Hsp40-type cochaperones of the ER: affinity for BiP. SPR analysis was carried out with immobilized
ERj3 (A), ERj3J (B), ERj5 (C), ERj5J (D), and ERj4 (E) and recombinant mouse BiP as described in Experimental procedures. We note that for
all Hsp40s there was no interaction observed in the absence of ATP or when ATPcS was used instead of ATP (not shown). The calculated
affinities are given in Table 1. We note that prior to application of BiP, dissociation of previously applied BiP was allowed to reach completion
(not shown).
Pancreatic endoplasmic reticulum chaperone network A. Weitzmann et al.
5180 FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS
complex in the absence of free ATP ) i.e. when at
least BiP can be expected to be in the ADP form –
and this complex is dissociated in the presence of an
excess of free ATP.
In order to confirm this interpretation, an immobi-
lized antibody that recognizes native Grp170 was

employed. A fraction from the gel filtration chroma-
tography in the absence of ATP that contained
approximately stoichiometric amounts of both chaper-
ones was incubated with immobilized antibodies to
Grp170 either in the absence or in the presence of
ATP. Subsequently, the antibody-bound and unbound
proteins were analyzed by SDS ⁄ PAGE and protein
staining (Fig. 6C, lanes 2 and 3 versus lanes 4 and 5).
The antibody to Grp170 coimmunoprecipitated BiP
more efficiently in the absence than in the presence of
ATP; that is, a significant amount of BiP remained in
the unbound fraction in the presence of ATP (Fig. 6C,
lane 3). Thus, the two chaperones are indeed able to
form a stable complex in the absence of ATP.
In the next experiment, the ability of Grp170 to
interact with Hsp40 was examined. BiP served as an
internal control for this experiment. In order to keep
the two Hsp70 chaperones from forming a complex,
ATP was present. A mixture of both Hsp70-type chap-
erones was incubated with an immobilized J-domain
(ERj1J). Subsequently, the J-domain-bound and
unbound proteins were analyzed by SDS ⁄ PAGE and
protein staining (Fig. 6D, lane 4 versus lane 2). As
expected, BiP was efficiently bound by the immobilized
J-domain. In contrast, Grp170 was not bound by the
immobilized J-domain, i.e. remained in the unbound
fraction (Fig. 6D, lane 2). Thus, in contrast to BiP,
Grp170 appears to be unable to form a stable complex
with Hsp40-type proteins.
Grp170 has a low basal ATPase activity that was

hardly stimulated by ERj1J and serves as a nucleotide
exchange factor for BiP in the presence of ERj1 [42]. In
order to analyze the nuleotide exchange activity of
Grp170 in the presence of the other ERjs, steady-state
A
E
I
B
F
J
C
G
K
D
H
L
Fig. 5. Functional characterization of Hsp40-type cochaperones and nucleotide exchange factors of the ER: effect on the ATPase activity of
BiP. ATP hydrolysis assays were carried out under steady-state conditions as described in Experimental procedures. The concentrations
were: ATP, 500 l
M; Sil1, 2 lM; BiP and Kar2p, 2 lM; Hsp40, 2 lM; and Grp170, 0.25 lM.
A. Weitzmann et al. Pancreatic endoplasmic reticulum chaperone network
FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5181
ATPase assays were carried out that involved BiP and
Grp170 ) in a physiological ratio ) plus Hsp40
(Fig. 5A–E; Table 1). The established nucleotide
exchange factor Sil1 ) at about two-fold molar
excess ) served as a positive control in these experi-
ments (Fig. 5F–J). Under conditions of stimulation of
BiP’s ATPase activity by any ER-resident Hsp40,
Grp170 led to further acceleration of ATP hydrolysis.

Thus Grp170 can serve as a nucleotide exchange factor
for BiP after stimulation of BiP by any ER-resident
Hsp40. Grp170 was more efficient than Sil1 in this
respect. We note that Sil1 had previously been shown
to stimulate the ATPase activity of BiP in the presence
of ERj4 under slightly different conditions [40]. There-
fore, the apparent lack of nucleotide exchange factor
activity of Sil1 in the presence of ERj3, Erj4 and ERj5
in our experiments should not be taken as an indication
of a specialized function of Sil1 (Fig. 5H–J; Table 1).
However, the data point to the fact that the two nucleo-
tide exchange factors have different efficiencies.
Both ERj1 and ERj2
⁄
Sec63 as well as Grp170
functionally interact with the yeast BiP ortholog
Kar2p
The yeast ortholog of BiP that is termed Kar2p was
observed to be unable to substitute for BiP in facili-
tating Sec61 channel gating in canine pancreatic
150
AB
CD
- ATP
Grp170
Grp170
+ BiP
fraction number
BiP
BiP

+ ATP
100
50
0
0 5 10 15 20
fraction number
0 5 10 15 20
Protein (arbitrary units)
450
300
150
0
Protein (arbitrary units)
Fig. 6. Characterization of Grp170: functional interactions. Superose 6 gel filtration was carried out as described in Experimental procedures
in the absence (A) and presence (B) of ATP. Fractions were collected and subjected to SDS ⁄ PAGE and subsequent staining with Coomassie
Brilliant Blue. Staining intensity was quantified by densitometry. Grp170 (open squares) and BiP (filled circles) were identified as such by
western blotting. An aliquot of fraction 5 of the gel filtration in the absence of ATP (termed input and shown in lane 1) was incubated with
immobilized antibodies to Grp170 in the absence or presence of ATP as indicated (C). The unbound (lanes 2 and 3) and bound (lanes 4 and
5) proteins were collected and subjected to SDS ⁄ PAGE and subsequent staining with Coomassie Brilliant Blue. An aliquot of an ATP eluate
of ATP–agarose chromatography was incubated with GSH–Sepharose (– ERj1J) or immobilized ERj1J (+ ERj1J) in the presence of ATP (D).
Subsequently, unbound (lanes 1 and 2) and bound (lanes 3 and 4) material were separated by centrifugation, and analyzed by SDS ⁄ PAGE
and protein staining with Coomassie Brilliant Blue. The protein ladder was run on the same gel (lane 5).
Pancreatic endoplasmic reticulum chaperone network A. Weitzmann et al.
5182 FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS
microsomes [18]. In fact, it even had a dominant nega-
tive effect on BiP in these experiments. Furthermore,
this gating activity of BiP was shown to involve an
unidentified resident ER Hsp40 [18]. In analogy to the
situation in yeast, the respective Hsp40 is expected to
be a membrane protein in pancreatic microsomes. As

lack of interchangeability of various Hsp70s has been
observed previously [48,49], it seemed reasonable that
the failure of Kar2p to support a complete ATPase
cycle in concert with one of the two most likely candi-
date Hsp40s in channel gating, ERj1 and ERj2, or the
major pancreatic nucleotide exchange factor, Grp170,
was responsible for the effects of Kar2p in channel gat-
ing in mammalian microsomes. At first, we addressed
the question of whether Kar2p functionally interacts
with the two relevant J-domains. Kar2p was stimulated
in its ATPase activity by the two J-domains to an
extent that is comparable to their stimulation of BiP
(Fig. 5K,L). The stimulation by ERj1J was 3-fold and
that by ERj2J was 2.5-fold, as compared to 5.2-fold
and 3.5-fold (Table 1). Next, we deterrmined whether
Grp170 functionally interacts with Kar2p after stimula-
tion of its ATPase activity by ERj1J or ERj2J. Grp170
stimulated the ATPase activity of Kar2p to an extent
that is comparable to the stimulation of BiP
(Fig. 5K,L). The stimulation by Grp170 in the presence
of ERj1J was 2.4-fold and that of ERj2J was 2.2-fold,
as compared to 5.6-fold and 5.7-fold (Table 1). The
observed differences appear to be too small to provide
an explanation for the inability of Kar2p to substitute
for BiP in Sec61 gating in mammalian microsomes.
Thus, lack of interchangeability between BiP and
Kar2p at the level of the Hsp40s ERj1 and ERj2 and at
the level of the nucleotide exchange factor Grp170 does
not appear to be responsible for the observed effect of
Kar2p in channel gating experiments [18].

Discussion
The pancreatic network of ER-luminal chaperones
under steady-state conditions
From the concentrations of the various chaperones
and cochaperones in the lumen of canine pancreatic
rough microsomes (RMs), one can extrapolate to the
situation in the corresponding rough ER (Table 1,
Fig. 7). It has to be taken into account that during
preparation of microsomes, about 50% of the luminal
content is lost into the postribosomal supernatant and
that the luminal volume of the microsomes is only a
minor fraction of the total volume of the microsomal
suspension (we estimate 1 : 500). Taken together, we
estimate that in the rough ER, the concentrations of
the most abundant chaperones such as BiP and ERj5
or their J-domains, such as in the case of ERj2 ⁄ Sec63,
are in the low millimolar range. Furthermore, the total
amounts of Hsp70 and Hsp40 proteins in the ER
lumen (Table 1) and the observed affinities (Table 1)
allow one to conclude that, in principle, all J-domains
can be associated with BiP at any given time. In real-
ity, however, a large proportion of BiP will be engaged
with polypeptide substrates. Therefore, one can assume
that under these steady-state conditions, the deter-
mined affinities become relevant. On the basis of the
analogies with yeast, and the facts that human ERj1
can complement deletion of the SEC63 gene in yeast,
and that ERj4 appears to be absent from pancreatic
rough ER under nonstress conditions, the two Hsp40s
ERj1

0.18mM
ERj2
0.99mM
ERj3
0.29mM*
ERj4
0mM*
ERj5
2mM*
0.12µM 5µM 3.6 µM 6µM 0.45µM
UPR
______________________________
BiP
5mM*
sensors:
PERK
IRE1
ATF6
Grp170
0.6mM*
Sil1
0.005mM
Fig. 7. The BiP network in the pancreatic ER. The estimated ER-luminal concentration and the measured affinities are indicated. The
observed concentrations for RMs (Table 1) were corrected for the facts that about 50% of the luminal proteins leak out of the organelle dur-
ing tissue homogenization and that the inner volume of the RM is small as compared to the total volume of the RM suspension (approxi-
mately 1 : 500), in order to estimate the concentration of the luminal proteins in the ER of pancreatic cells. UPR, unfolded protein response;
*UPR-inducible.
A. Weitzmann et al. Pancreatic endoplasmic reticulum chaperone network
FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5183
ERj1 and ERj2 ⁄ Sec63 appear to be the prime candi-

dates for alternatively cooperating with BiP in protein
transport. This may explain why disruption of
SEC63 ⁄ ERJ2 in autosomal dominant polycystic liver
disease is not lethal, in contrast to the situation in
yeast [44]. Grp170 was characterized as an alternative
nucleotide exchange factor for BiP [42]. This is in per-
fect agreement with the fact that disruption of SIL1 ⁄
BAP in humans and mice does not cause lethality
[45,46,50]. Thus, as in yeast, the presence of the addi-
tional nucleotide exchange factor Grp170 may com-
pensate for the loss of Sil1.
The network of ER-luminal chaperones under
stress conditions
When misfolded proteins accumulate in the ER, vari-
ous signal transduction pathways are activated that
increase the biosynthetic capacity and decrease the bio-
synthetic burden of the ER. This phenomenon is
termed the unfolded protein response. Most of the
members of the chaperone network discussed here are
under control of the unfolded protein response (indi-
cated by an asterisk in Fig. 7). Therefore, one would
expect ERj4 to be present in pancreatic microsomes
under stress conditions. Furthermore, the GRP170 gene
would be expected to be overexpressed after disruption
of SIL1 ⁄ BAP in humans and mice. Thus, overexpres-
sion of GRP170 may compensate for the loss of Sil1 in
most tissues. However, for unknown reasons, this does
not seem to work for certain areas of the cerebellum in
patients suffering from Marinesco–Sjo
¨

gren syndrome
[45,46] and in the so-called woozy mice [50].
The ATPase cycle of BiP
In principle, BiP’s ATPase cycle follows the well-estab-
lished, paradigmatic functional cycle of DnaK [51].
Briefly, BiP–ATP has a low affinity for polypeptide
substrates. Proteins with BiP-reactive J-domains have a
high affinity for BiP–ATP and can therefore bind to
the underside of the ATP-binding cleft. Owing to their
additional domains, the Hsp40s ERj3, ERj4 and ERj5
may be able to bind polypeptide substrates and deliver
them to peptide-binding domains of BiP in the course
of their interaction with BiP. In contrast, in the case of
ERj1 and ERj2, BiP appears to be recruited to the
substrate polypeptides by spatial proximity to the
Sec61 complex and ribosomes, respectively. In any
case, interaction of the J-domain with the ATP-binding
cleft triggers ATP hydrolysis and a subsequent confor-
mational change in the peptide-binding domain.
Apparently, in the in vitro analysis in the absence of
polypeptide substrates, this leads to reversible trapping
of the J-domain or neighboring parts of Hsp40 in the
peptide-binding domain. In the presence of BiP sub-
strates, Hsp40s dissociate from BiP, and the polypep-
tide substrates are trapped by BiP. Next, BiP-bound
ADP is exchanged for ATP, and the above-mentioned
conformational change in the peptide-binding domain
is reversed. Substrate is released, and BiP is ready for
the next round of the cycle. Typically, ADP–ATP
exchange is catalyzed by a nucleotide exchange factor,

such as Grp170 in the pancreatic ER, that has a high
affinity for BiP–ADP. We note that our observation of
a stable complex of Grp170 and BiP is perfectly in line
with previous observations of chaperone complexes in
the ER lumen [52,53].
Experimental procedures
Materials
The protein ladder (10–200 kDa) was obtained from Life
Technologies (Grand Island, NY, USA). ATP-C
8
-agarose,
thrombin and peroxidase conjugate of goat anti-(rabbit IgG)
serum were obtained from Sigma Chemical Company, Tauf-
kirchen, Germany). [
32
P]ATP, ATP, GSH–Sepharose 4 Fast
Flow, protein A–Sepharose and protein G–Sepharose 4 Fast
Flow, Superose 6B, X-ray films and the enhanced chemilu-
minescence (ECL) were obtained from GE Healthcare (Frei-
burg, Germany). Poly(vinylidene difluoride) membranes and
Centricon devices were obtained from Millipore (Schwal-
bach, Germany). Chaps was obtained from Calbiochem
(Schwalbach, Germany). Hepes and Coomassie Brilliant
Blue were purchased from Serva (Heidelberg, Germany).
Purification of proteins from dog pancreas
Dog pancreas microsomes were prepared as previously
described [13]. The microsomes were stripped with respect
to ribosomes according to published procedures [11]. After
reisolation of microsomes by centrifugation, the pellets were
resuspended in extraction buffer (20 mm Hepes ⁄ KOH,

pH 7.5, 400 m m KCl, 1 mm EDTA, 1.5 mm MgCl
2
,2mm
dithiothreitol, 15% w ⁄ v glycerol, 0.65% w ⁄ v Chaps), result-
ing in a crude extract. Typically, the ribosomes were
pelleted by centrifugation for 30 min at 2 °C and 240 000 g
in a Beckman TLA 100.3 rotor. Purification of ATP-bind-
ing proteins was carried out on ATP-C
8
-agarose as
described previously [13]. Where indicated, the ATP eluate
was concentrated in Centricon devices and subjected
to A
¨
kta chromatography in a Superose 6B column
(16 · 500 mm) (GE Healthcare). The running buffer was
identical to the extraction buffer, except that glycerol was
omitted, KCl was reduced to 200 mm, and 4 m m Mg-ATP
was added where indicated.
Pancreatic endoplasmic reticulum chaperone network A. Weitzmann et al.
5184 FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS
Purification of recombinant proteins
Purification of recombinant GST and GST hybrid proteins
was carried out on GSH-Sepharose 4 Fast Flow in a man-
ner similar to that described for purification of a GST–
Sec63J hybrid [21]. Sil1 was purified after cleavage of
GST-Sil1 hybrid with thrombin. Purification of His-tagged
murine BiP is described elsewhere [33].
Antibodies
Antibodies against Sil1 were raised after cleavage of GST-

Sil1 hybrid with thrombin. Antibodies against Grp170
(LAVMSVDLGSEC), ERj4 (TVQTENRFHGSSKHC) and
ERj5 (CLRNQGKRNKDEL) were raised against the indi-
cated peptides plus an additional N-terminal or C-terminal
cysteine as previously described [21]. The antipeptide anti-
bodies were affinity purified and immobilized on a mixture
of protein A–Sepharose and protein G–Sepharose as previ-
ously described [21]. Immunoaffinity purification was car-
ried out in the above-defined gel filtration buffer as
previously described [21].
Quantitation of proteins in dog pancreas
microsomes
The amount of protein present in the band of a gel of a
GST hybrid preparation was determined by comparison
with protein standards that were separated on the same gel
and were stained with Coomassie Brilliant Blue simulta-
neously. Subsequently, an aliquot of the same sample of
purified GST hybrid protein was separated on the same gel
together with increasing amounts of microsomes [21]. The
known amount of purified protein served as a standard for
the western blot signals, as determined by luminescence and
densitometry of the X-ray films.
Pull-down assay
GST or a GST hybrid was immobilized on GSH–Sepha-
rose. The supernatant, derived from the detergent extract of
microsomes, was diluted by the addition of one volume of
salt-free extraction buffer and applied to the immobilized
proteins in the absence or in the presence of ATP (2 mm).
The columns were washed with application buffer [21]. The
bound proteins were eluted and subjected to SDS ⁄ PAGE,

and subsequent staining with Coomassie Brilliant Blue.
SPR spectroscopy
SPR spectroscopy was carried out in a BIAlite upgrade
system as previously described [21]. Briefly, monoclonal
goat anti-GST serum (BIACORE, Freiburg, Germany)
was immobilized on a CM5 research grade sensor chip
(BIACORE) by amine coupling according to the manufac-
turer’s protocol. The chip was equilibrated with application
buffer supplemented with ATP (final concentration: 2 mm)
and Tween-20 (final concentration: 0.1%), termed running
buffer (flow rate: 15 lLÆmin
)1
). GST–J-domain hybrid was
bound to the immobilized antibodies in the measuring cell.
Similarly immobilized GST in the reference cell served as
a negative control. Subsequently, solutions containing
increasing concentrations of purified BiP (0.05–12 lm) were
passed over the chip in the presence of ATP. Each BiP
application was followed by application of running buffer.
The analysis was carried out employing the BIA evaluation
software version 2.2.4 (BIACORE).
Steady-state ATPase assay
Steady-state assays were incubated for 60 min at 37 °Cin
40 mm Hepes ⁄ KOH (pH 7.4), 25 mm KCl, and 2.5 mm
MgCl
2
[21]. The standard protein concentrations were: Sil1,
2 lm;BiP,2lm;Hsp40,2lm; and Grp170, 0.25 lm.Reac-
tions were started by adding 5% (v⁄ v) of an ATP cocktail that
contained 0.1 lCi of [

32
P]ATP[cP] (4500 CiÆmmol
)1
)per
100 lLplus10mm unlabeled ATP. Aliquots were removed
from the assay at the indicated times and quenched with an
equal volume of 60 mm EDTA. The samples were analyzed by
TLC on polyethyleneimide–cellulose in 1 m formiate and
0.5 m LiCl. ATP hydrolysis was calculated after the TLC
plates were analyzed by phosphorimaging (imagequant
software 5.1; Molecular Dynamics, Krefeld, Germany). We
note that there was a certain variation in the basal ATP hydro-
lysis rates of BiP, depending on the batch of [
32
P]ATP[cP].
Therefore, data from one experiment were compared.
Acknowledgements
We wish to thank Drs Linda Hendershot and Giannis
Spyrou for providing us with antibodies directed
against Sill, Erj4, and Erj5, respectively. Furthermore,
we are grateful to Drs Linda Hendershot and Greg
Blatch for stimulating discussions. This work was sup-
ported by SFB530.
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