Báo cáo khoa học: CHIP participates in protein triage decisions by preferentially ubiquitinating Hsp70-bound substrates pdf

CHIP participates in protein triage decisions by
preferentially ubiquitinating Hsp70-bound substrates
Marta Stankiewicz
1
, Rainer Nikolay
1,
*, Vladimir Rybin
2
and Matthias P. Mayer
1
1 Zentrum fu
¨
r Molekulare Biologie der Universita
¨
t Heidelberg (ZMBH), DKFZ–ZMBH Alliance, Heidelberg, Germany
2 European Molecular Biology Laboratory, Heidelberg, Germany
Introduction
CHIP consists of an N-terminal TPR (tetratricopeptide
repeat) domain, which binds to the C-terminal EEVD
motif that is present in all cytosolic Hsp70 and Hsp90
chaperones, a central helical domain, which is essential
Keywords
chaperones; Hsp70; Hsp90; protein triage;
ubiquitination
Correspondence
M. P. Mayer, Zentrum fu
¨
r Molekulare
Biologie der Universita
¨
t Heidelberg (ZMBH),

DKFZ–ZMBH Alliance, Im Neuenheimer Feld
282, 69120 Heidelberg, Germany
Fax: +49 6221 545894
Tel: +49 6221 546829
E-mail:
*Present address
Biochemisches Institut der Universita
¨
t
Zu
¨
rich, Winterthurerstrasse 190, 8057
Zu
¨
rich, Switzerland
(Received 3 May 2010, revised 8 June
2010, accepted 14 June 2010)
doi:10.1111/j.1742-4658.2010.07737.x
The E3 ubiquitin ligase CHIP (C-terminus of Hsc70-interacting protein) is
believed to be a central player in the cellular triage decision, as it links the
molecular chaperones Hsp70 ⁄ Hsc70 and Hsp90 to the ubiquitin proteaso-
mal degradation pathway. To better understand the decision process, we
determined the affinity of CHIP for Hsp70 and Hsp90 using isothermal
titration calorimetry. We analyzed the influence of CHIP on the ATPase
cycles of both chaperones in the presence of co-chaperones and a substrate,
and determined the ubiquitination efficacy of CHIP in the presence of the
chaperones. We found that CHIP has a sixfold higher affinity for Hsp90
compared with Hsc70. CHIP had no influence on ADP dissociation or
ATP association, but reduced the Hsp70 cochaperone Hdj1-stimulated sin-
gle-turnover ATPase rates of Hsc70 and Hsp70. CHIP did not influence

the ATPase cycle of Hsp90 in the absence of co-chaperones or in the pres-
ence of the Hsp90 cochaperones Aha1 or p23. Polyubiquitination of heat-
denatured luciferase and the native substrate p53 was much more efficient
in the presence of Hsc70 and Hdj1 than in the presence of Hsp90, indicat-
ing that CHIP preferentially ubiquitinates Hsp70-bound substrates.
Structured digital abstract
l
MINT-7904367: CHIP (uniprotkb:Q9UNE7) and HSP 90-beta (uniprotkb:P08238) physically
interact (
MI:0915)bymolecular sieving (MI:0071)
l
MINT-7904785: HSP 90-beta (uniprotkb:P08238) and p23 (uniprotkb:Q15185) bind
(
MI:0407)bymolecular sieving (MI:0071)
l
MINT-7904047: CHIP (uniprotkb:Q9UNE7), HSP 90-beta (uniprotkb:P08238) and p23 (uni-
protkb:
Q15185) physically interact (MI:0915)bymolecular sieving (MI:0071)
l
MINT-7903424: Alpha-lactalbumin (uniprotkb:P00711), HSP70 (uniprotkb:P08107) and
CHIP (uniprotkb:
Q9UNE7) physically interact (MI:0915)bymolecular sieving (MI:0071)
l
MINT-7903354: CHIP (uniprotkb:Q9UNE7) and HSC70 (uniprotkb:P11142) bind (MI:0407)
by isothermal titration calorimetry (
MI:0065)
l
MINT-7903373: CHIP (uniprotkb:Q9UNE7) and HSP90-beta (uniprotkb:P08238) bind
(
MI:0407)byisothermal titration calorimetry (MI:0065)

Abbreviations
CHIP, C-terminus of Hsc70-interacting protein; Hsp70, 70 kDa heat shock protein; Hsp90, 90 kDa heat shock protein; Hsc70, 70 kDa heat
shock cognate; ITC, isothermal titration calorimetry; MABA-ADP ⁄ MABA-ATP, N
8
-(4-N¢-methylanthraniloylaminobutyl)-8-aminoadenosine
5¢-di ⁄ triphosphate; RCMLA, reduced carboxymethylated a-lactalbumin; TPR, tetratricopeptide repeat.
FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS 3353
for dimerization, and a C-terminal U-box domain,
which is responsible for interaction with E2 ubiquitin-
conjugating enzymes [1,2]. This interaction with molec-
ular chaperones suggests that CHIP is involved in the
triage decision [3–5]. Hsp70 chaperones are essential
components of the cellular quality control network,
interacting with virtually all misfolded proteins, pre-
venting their aggregation and assisting their refolding
into the native state [6–8]. Hsp90 chaperones have also
been shown to bind to misfolded proteins [9], but their
main essential function in all eukaryotic cells is
believed to be interaction with a large number of regu-
latory proteins, called client proteins, including recep-
tors, protein kinases and transcription factors [10–12].
The question therefore arises as to whether CHIP pref-
erentially interacts with Hsp70 to mark misfolded
proteins, which cannot be refolded to the native
state, for degradation, or whether it assumes a more
regulatory role by interacting mainly with Hsp90 to
ubiquitinate signaling proteins.
It has been shown in vitro and in cell culture that sev-
eral Hsp90 clients are ubiquitinated in a CHIP-depen-
dent manner, and that this ubiquitination depends on

CHIP’s U-box domain [13–17]. In some cases, CHIP
appears to directly bind its substrates and ubiquitinate
them in a chaperone-independent manner [18–20]. Such
specific substrate recognition is a typical feature of E3
ubiquitin ligases; however, the majority of CHIP’s sub-
strates described so far are bona fide Hsp70 and Hsp90
clients, and their degradation depends on the presence
of the chaperones [13,21–23]. Among the CHIP
substrates are many regulatory proteins that are ubiqui-
tinated and degraded even in the presence of an Hsp90
inhibitor such as geldanamycin [21].
For most if not all functions, Hsp90 cooperates with
Hsp70 in a chaperone cycle, which was first proposed
for steroid hormone receptors and involves a number
of co-chaperones [24,25]. Steroid hormone receptors
first interact with Hsp40 and Hsp70. The dimeric pro-
tein Hop (Hsp70-Hsp90 organizing Protein), which has
separate TPR domains for binding to Hsp70 (TPR1)
and Hsp90 (TPR2a), assembles the early client com-
plex with Hsp70 and Hsp90. Hop and Hsp70 are then
replaced by p23 and a TPR domain-containing pept-
idyl-prolyl-cis ⁄ trans-isomerase (e.g. the 51 and 52 kDa
FK506-binding proteins FKBP51 or FKBP52). The
mature complex decays with a half life of approxi-
mately 5 min, and the hormone receptor re-enters the
cycle by binding to Hsp40 and Hsp70. As CHIP can
interact with Hsp70 and Hsp90, it is not clear whether
ubiquitination of the chaperone substrate occurs while
the substrate is bound to Hsp70 or Hsp90. Another
intriguing question is how the triage decision is made.

As CHIP is a TPR-containing co-chaperone, it com-
petes with numerous other TPR-containing co-chaper-
ones for binding to Hsp70 and Hsp90 [26].
Here we provide new insights into the triage decision
by assessing the physical interaction of CHIP with
Hsp70 ⁄ Hsc70 and Hsp90, and analyzing the functional
consequences for the chaperone substrates.
Results
CHIP–chaperone interaction and competition with
Hop
To determine how the triage decision is made, we first
addressed the question of protein affinities and cellular
concentrations. CHIP directs proteins to the degrada-
tion pathway, and Hop is an essential co-chaperone
for protein folding. Because they both interact with
the same C-terminal EEVD motif of Hsc70 and
Hsp90, we determined the affinities of CHIP for Hsc70
and Hsp90 using isothermal titration calorimetry
(ITC) ( Fig. 1A,B). We also investigated the interaction
of CHIP with heat shock-induced Hsp70, to determine
whether CHIP has a preference for this homolog to
enhance quality control processes during heat shock.
CHIP’s affinity for Hsp90 (K
D
= 0.38 ± 0.04 lm)
was approximately six times higher than that for
Hsc70 (K
D
= 2.3 ± 0.3 lm), and two and a half times
higher than that for Hsp70 (K

D
= 0.95 ± 0.01 lm).
The affinities of CHIP for Hsc70 and Hsp70 were in
the same range as the value measured for the Hop–
Hsc70 interaction (K
D
= 1.5 ± 0.2 lm) using surface
plasmon resonance spectroscopy [27]. In contrast,
for the interaction of Hop with Hsp90, a K
D
value
of 0.1 ± 0.02 lm was determined by surface plasmon
resonance spectroscopy, which is only one quarter of
the value measured here for the CHIP–Hsp90 interac-
tion. These results indicate that Hop and CHIP com-
pete efficiently with each other for binding to
Hsc70 ⁄ Hsp70 when the C-termini of Hsc70⁄ Hsp70 are
limiting when the concentration of Hsc70/Hsp70 is
lower than the combined concentration of CHIP and
Hop, but Hop appears to be at an advantage com-
pared to CHIP when binding to Hsp90.
The cellular concentrations of Hsc70⁄ Hsp70, Hsp90,
CHIP and Hop where determined by quantitative Wes-
tern blot using HEK293, a commonly used epithelial
cell line, and Jurkat cells, which are a model for acute
T-cell leukemia (Fig. 1C and Table 1). The values
determined for Hsc70 ⁄ Hsp70 (0.9 and 0.4% of total
protein for HEK293 and Jurkat cells, respectively) and
Hsp90 (0.6 and 0.8% of total protein for HEK293 and
Jurkat cells, respectively) were somewhat lower than

CHIP preferentially ubiquitinates Hsp70 substrates M. Stankiewicz et al.
3354 FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS
the values of 1–2% reported for other cell lines [27a].
For Hop, we determined a relative amount of 0.2% in
both cell lines. In contrast, the relative amount of
CHIP varied significantly, being 0.07% in HEK293
and only 0.01% in Jurkat cells. As the total protein
concentration was 154 mgÆmL
)1
in HEK293 cells [28]
and 127 mg ÆmL
)1
in Jurkat cells [29], the concentra-
tions of Hsc70 ⁄ Hsp70 are 20 and 7 lm, those of
Hsp90 are 11 and 12 lm, those of Hop are 5 and
4 lm, and those of CHIP are 3.1 and 0.4 lm in
HEK293 and Jurkat cells, respectively. Given these
concentrations of Hsc70, Hsp90, CHIP and Hop and
the K
D
values, approximately 8.5 and 1.4% of Hsc70
and 4.6 and 0.7% of Hsp90 molecules have CHIP
bound to their C-termini at any given moment in
HEK293 and Jurkat cells, respectively. As there are a
large number of TPR domain proteins in higher
eukaryotic cells, many of which bind to Hsp90 with a
similar affinity as CHIP and Hop do [30,31], the
amount of Hsp90 occupied by CHIP is probably much
lower than estimated above. Fewer TPR proteins have
been shown to bind Hsc70 ⁄ Hsp70 [26]. Therefore,

based on our affinity determination and quantitative
Western blots, the amount of Hsc70 occupied by
CHIP is estimated to be 1–9% (in Jurkat and HEK293
cells).
Influence of CHIP on substrate binding of Hsp70
RING and U-box E3 ligases do not transfer ubiquitin
themselves but generally bring substrates and E2
ubiquitin-conjugating enzymes in close proximity by
binding to both proteins. It has been shown that CHIP
has the ability to bind substrates [18–20]. If CHIP also
contacts substrates when bound to Hsp70, it might
increase the stability of the Hsp70–substrate complex,
thereby allowing more time for ubiquitin transfer by
the E2 enzyme.
We therefore assessed whether CHIP affects the
equilibrium dissociation constant (K
D
) or the dissocia-
tion rate constant (k
off
) of the Hsp70–substrate
complex by analyzing the formation of complexes of
Hsp70 with reduced carboxymethylated a-lactalbumin
Fig. 1. Interaction of CHIP with Hsc70 and
Hsp90 and in vivo concentrations of the
chaperones and co-chaperones. (A,B) Deter-
mination of the interaction parameters of
the CHIP–Hsc70 (A) and CHIP–Hsp90 (B)
complexes using isothermal titration calorim-
etry. (C) Quantitative immunoblot for deter-

mination of the in vivo concentrations of
Hsp70 ⁄ Hsc70, Hsp90, CHIP and Hop in
HEK293 and Jurkat cells. Various amounts
of purified protein (15–400 ng, left panels)
and cleared protein extracts (10–100 lg) of
HEK293 (middle panels) and Jurkat cells
(right panels), as indicated, were separated
by SDS ⁄ PAGE and analyzed by immunoblot-
ting with specific antisera. The upper bands
detected in vivo for CHIP and Hop most
likely represent phosphorylated variants of
the proteins [72,73].
Table 1. Relative amounts of CHIP, Hop, Hsp70 and Hsp90 in
HEK and Jurkat cells. The relative amounts of chaperone and
co-chaperones were determined by quantitative immunoblotting as
shown in Fig. 1C using purified proteins as standards.
Percentage of
total protein HEK Jurkat
CHIP 0.072 ± 0.014 0.01 ± 0.0002
Hop 0.20 ± 0.01 0.18 ± 0.0001
Hsp70 0.94 ± 0.001 0.38 ± 0.007
Hsp90 0.60 ± 0.01 0.80 ± 0.05
M. Stankiewicz et al. CHIP preferentially ubiquitinates Hsp70 substrates
FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS 3355
(RCMLA), a model chaperone substrate [32], in the
presence and absence of CHIP using gel filtration and
3
H-RCMLA (Fig. 2A,B). When Hsp70 was pre-incu-
bated with CHIP, the amount of RCMLA bound to
Hsp70 decreased by approximately 40% (Fig. 2B).

This was not observed with the CHIP-K30A variant,
which does not bind to the C-terminal EEVD motif of
Hsp70 and Hsp90 [21], suggesting that CHIP does not
compete for Hsp70’s substrate binding pocket but
affects the chaperone–substrate interaction in an indi-
rect way. This result clearly indicates that CHIP does
not prolong the half life of the high-affinity Hsp70–
chaperone complex.
To investigate substrate release, we chased the pre-
formed Hsp70–
3
H-RCMLA complexes with unlabeled
RCMLA in the absence and presence of CHIP. As
shown in Fig. 2C, CHIP did not influence substrate
release by Hsp70, suggesting that the decrease in
detectable Hsp70–RCMLA complex is due to a
decreased association rate. CHIP bound to the C-ter-
minus of Hsp70 possibly creates a steric hindrance to
substrate binding. We did not observe any direct inter-
action of CHIP with the chaperone substrate RCMLA,
suggesting that substrate binding by CHIP may be a
specific interaction limited to certain proteins. In con-
clusion, CHIP did not increase the half life of the
Hsp70–RCMLA complex by directly stabilizing the
chaperone–substrate interaction, but instead decreased
the amount of Hsp70-bound RCMLA by 40%.
Influence of CHIP on the ATPase cycle of Hsp70
As nucleotide exchange by Hsp70 is rate-limiting for
substrate release under physiological ATP concentra-
tions, CHIP could also affect the half-life of the

Hsp70–substrate complex by altering the ATPase cycle
of Hsp70 proteins. It has been reported that CHIP
decreases the ATPase rate stimulated by the J-domain
co-chaperones Hdj1 and Hdj2 but not the basal
ATPase rate under steady-state conditions [14,33]. In
the absence of a J-domain co-chaperone, c-phosphate
cleavage is rate-limiting in the ATPase cycle of Hsp70
proteins [34,35]. In the presence of a J-domain protein,
nucleotide exchange becomes rate-limiting [36,37]. The
CHIP-induced reduction of the Hdj1 ⁄ Hdj2-stimulated
ATPase rate of Hsc70 could therefore be caused by a
reduced nucleotide exchange, which in turn would
increase the dwell time of the substrate on the Hsp70
chaperone. To address this point, we analyzed the
influence of CHIP on ADP dissociation from and
ATP association with Hsc70 and Hsp70 using the
fluorescent nucleotide analogs N
8
-(4-N¢-methylanthra-
niloylaminobutyl)-8-aminoadenosine 5¢-di ⁄ triphosphate
(MABA-ADP ⁄ MABA-ATP) [38] and stopped-flow
instrumentation. To measure the basal ADP dissocia-
tion rate, Hsc70 or Hsp70 were pre-incubated with
MABA-ADP in the absence or presence of a 20-fold
excess of CHIP, and subsequently mixed with an
Fig. 2. CHIP reduces the affinity of Hsp70 for a model substrate
without affecting the dissociation rate. (A) Size-exclusion chroma-
tography of
3
H-RCMLA (reduced carboxymethylated a-lactalbumin)

after pre-incubation in the absence or presence of Hsp70 and CHIP
as indicated. (B) Quantification for the size-exclusion chromatogra-
phy experiments shown in (A).
3
H-RCMLA was pre-incubated with
the indicated proteins. The amount of radioactivity in elution volume
9–11.5 mL, in which the RCMLA–Hsp70 complex elutes, is shown
relative to the radioactivity in elution volume 12–15.5 mL, in which
free RCMLA elutes. (C) Dissociation of the RCMLA–Hsp70 com-
plex.
3
H-RCMLA was pre-incubated with Hsp70 before addition of
CHIP where indicated. At time point 0, a fivefold excess of unla-
beled RCMLA was added. The complex was analyzed by size-exclu-
sion chromatography at various time points. The dissociation rate
constants (k
off
) were determined by fitting a single exponential
decay function to the data. The inset shows the dissociation rate
constants in the absence and presence of CHIP (mean ± SEM of
two independent determinations).
CHIP preferentially ubiquitinates Hsp70 substrates M. Stankiewicz et al.
3356 FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS
excess of ATP. As shown in Fig. 3A, CHIP had no
influence on the basal ADP dissociation rates of Hsc70
and Hsp70. These results did not explain the CHIP-
mediated decrease in the Hdj1⁄ Hdj2-stimulated steady-
state ATPase rate.
In vivo nucleotide exchange factors such as Bag-1
accelerate ADP dissociation by Hsc70 and Hsp70

[37,39]. It has been reported that CHIP and Bag-1
interact with each other [40]. We therefore determined
whether CHIP could influence Bag-1-stimulated nucle-
otide exchange. To address this question, we pre-incu-
bated Hsc70 ⁄ Hsp70 with MABA-ADP in the absence
and presence of a large excess of CHIP, and subse-
quently rapidly mixed the reaction mixture with Bag-1
and an excess of ATP. As expected, Bag-1 stimulated
the ADP dissociation rate by approximately 20-fold at
stoichiometic concentrations. Even a large excess of
CHIP only slightly decreased the Bag-1-stimulated
ADP dissociation rate of Hsc70 and Hsp70 (Fig. 3A).
No effect of CHIP on the Bag-1-stimulated ADP dis-
sociation rate was observed when CHIP was added
together with Bag-1 instead of pre-incubated with the
Hsp70 protein (data not shown). Therefore, the
reported interaction of CHIP and Bag-1 has no strik-
ing effect on the nucleotide release function of Bag-1.
To analyze the second step of nucleotide exchange,
ATP association, we pre-incubated Hsc70 or Hsp70 in
the absence and presence of Bag-1 and a 20-fold excess
of CHIP, and subsequently mixed the reaction mixture
with MABA-ATP. As shown in Fig. 3B,C, CHIP did
not slow down ATP association significantly in the
absence or presence of Bag-1. Instead we observed a
slight increase in ATP association rate for Hsc70 in
the presence of CHIP.
As neither ADP dissociation nor ATP association are
negatively affected by CHIP, the reduction in the
ATPase activity must be due to an effect on c-phos-

phate cleavage. To verify this hypothesis, we performed
single-turnover ATPase experiments. The basal ATPase
rate of Hsp70 proteins is very low but can be stimulated
by a J-domain co-chaperone at high concentrations
(> 10-fold). As shown in Fig. 3D, high concentrations
of CHIP had no effect on the basal single-turnover
ATPase rate but decreased the Hdj1-stimulated ATPase
Fig. 3. CHIP affects Hdj1-stimulated c-phosphate cleavage by
Hsc70 ⁄ Hsp70 but not nucleotide exchange. (A) MABA-ADP dissoci-
ation rates of Hsc70 and Hsp70 in the absence and presence of
CHIP and Bag-1. (B) Fluorescence traces of MABA-ATP association
with Hsc70 in the absence and presence of CHIP and Bag-1. (C)
Association rates for MABA-ATP in the absence and presence of
Bag-1 and CHIP. The columns show the rates for the fast phase
(k
1
) and the slow phase (k
2
) of a fit of a two-phase exponential
equation to the traces in (B) and additional data. (D) Single-turnover
ATPase rates of Hsc70 and Hsp70 in the absence and presence of
Hdj1 and CHIP as indicated.
M. Stankiewicz et al. CHIP preferentially ubiquitinates Hsp70 substrates
FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS 3357
rate, consistent with previous steady-state ATPase data
[14,33]. Taken together, we found no evidence that
CHIP influences the chaperone cycle of Hsp70 proteins
to prolong the life-time of the substrate–Hsp70–CHIP
complex and thereby to increase the possibility of
recruitment of the E2 ubiquitin-conjugating enzyme and

ubiquitination. In contrast, CHIP decreased the
Hdj1-triggered c-phosphate cleavage, thereby decelerat-
ing transition from the low-affinity to the high-affinity
substrate-binding state.
Influence of CHIP on the ATPase activity and
co-chaperone binding of Hsp90
We next determined whether CHIP influences the
ATPase cycle of Hsp90 in order to increase the possibil-
ity of ubiquitination of an Hsp90-bound client protein.
We performed steady-state ATPase assays of Hsp90 in
the absence and presence of CHIP, and in the absence
and presence of Aha1 and p23, two co-chaperones that
are known to influence the ATPase activity of Hsp90.
We obtained a value of 1.2 ± 0.1 · 10
)3
s
)1
for the
basal ATPase activity of Hsp90. This rate was stimu-
lated fivefold by a threefold excess of Aha1 over Hsp90,
and inhibited to 50% of the basal rate by a tenfold
excess of p23, consistent with published data [41,42]. As
shown in Fig. 4, CHIP did not significantly affect the
basal ATPase rate of Hsp90, and also had no influence
on the Aha1-stimulated or p23-inhibited rate, even at a
tenfold excess over Hsp90.
A previous study suggested that p23 competes with
CHIP for binding to Hsp90 [13]. As CHIP did not
reduce the inhibitory effect of p23 on the ATPase
activity of Hsp90, we determined whether this is due

to the inability of CHIP to bind to Hsp90 in the pres-
ence of p23. We therefore incubated Hsp90 with CHIP
and p23 and used gel filtration to analyze the com-
plexes formed. As evident from Figs 5A,B and S1,
CHIP forms a stable complex with Hsp90 and p23 and
does not prevent p23 binding to Hsp90. In contrast,
Hop reduced binding of p23 to Hsp90, consistent with
previous observations [43]. Aha1 also reduced binding
of p23 to Hsp90, and CHIP could not reverse this
effect of Aha1. Taken together, CHIP did not reduce
the ATP hydrolysis rates of Hsp90. As ATP hydrolysis
leads to substrate release [44], CHIP should not
increase the half-life of Hsp90–client complexes.
Unfolded proteins are more efficiently
ubiquitinated in the presence of the Hsp70 system
As CHIP interacts with both Hsp70 and Hsp90 and
the interaction is mutually exclusive, we wished to
directly compare the two chaperone systems in terms
of their influence on the efficiency of CHIP-mediated
ubiquitination of a substrate. It has already been
shown, that both systems are able to support ubiqui-
tination in vitro, but quantitative time-resolved ubiq-
uitination experiments are necessary to compare their
relative efficiency. We pre-incubated the chaperone
substrate firefly luciferase in the presence of various
concentrations of Hsc70 plus Hdj1 or Hsp90 at
43 °C, and subsequently shifted the temperature to
30 °C before adding CHIP, the E2 enzyme UbcH5c,
the E1 ubiquitin-activating enzyme and ubiquitin. In
the presence of Hsc70 and Hdj1, ubiquitination was

very efficient even at low chaperone:luciferase ratios
Fig. 4. CHIP has no influence on the ATPase rate of Hsp90. (A)
Steady-state ATPase rate of Hsp90 in the absence or presence of
the indicated concentrations of CHIP. (B) Steady-state ATPase rate
of Hsp90 in the absence or presence of the indicated concentra-
tions of p23 and CHIP. (C) Steady-state ATPase rate of Hsp90 in
the absence or presence of the indicated concentrations of Aha1
and CHIP.
CHIP preferentially ubiquitinates Hsp70 substrates M. Stankiewicz et al.
3358 FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS
(1 : 1), but not in the absence of Hdj1 (Fig. 6A,B, left
panel). In contrast, ubiquitination in the presence of
Hsp90 was not very efficient and required high con-
centrations of chaperone. This indicates that many
more substrate molecules can be successfully ubiquiti-
nated per one Hsc70 ⁄ CHIP complex than per one
Hsp90 ⁄ CHIP complex. Time-resolved experiments
also showed that CHIP-mediated polyubiquitination
was faster in the presence of the Hsc70 ⁄ Hdj1 system
than in the presence of Hsp90 (Fig. 6B, right panel).
Interestingly, when we used the lysine-free variant of
ubiquitin (Ubi-K0) to prevent polyubiquitination, we
also detected multiple bands of luciferase in the pres-
ence of Hsc70 and Hsp90, indicating that both chap-
erones allow attachment of ubiquitin to several
lysines of luciferase. These data suggest that, several
lysines of the substrate are modified even in the pres-
ence of wild-type ubiquitin. As multiple bands of
ubiquitinated luciferase are already visible at the
20 min time point in the case of wild-type ubiquitin

but appear later in the case of the K0 ubiquitin vari-
ant, polyubiquitination may occur in the presence of
Hsp70 with a certain processivity, or alternatively the
lysines in ubiquitin (presumably Lys48) are better
substrates for ubiquitination than lysines in the sub-
strate.
In addition, the co-chaperones of Hsp70 regulate the
reaction in a dynamic manner. Hdj1 strongly enhanced
ubiquitination, as mentioned above (Fig. 6A), but
Bag-1 reduced the ubiquitination efficacy (Fig. 6C, left
panels). In contrast, neither p23 nor Aha1 had an
impact on the basal Hsp90-dependent ubiquitination
(Fig. 6C, right panels). The presence of Hop reduced
the ubiquitination of luciferase for both Hsc70 and
Hsp90; however, the effect was observed only after
shorter time periods, and polyubiquitinated species
accumulate after longer time periods, despite increas-
ing Hop concentrations (Fig. 6D). Both systems gener-
ate substrates with multiple ubiquitinated sites, as
shown for the reaction using a lysine-free ubiquitin
mutant (Fig. 6B). However, unfolded proteins are
more efficiently ubiquitinated in the presence of the
Hsp70 system.
Ubiquitination of a native chaperone substrate
protein
Hsp70 and Hsp90 not only interact with misfolded
proteins but also with native or near-native proteins.
To investigate CHIP-mediated ubiquitination of a
native protein substrate, we chose the tumor suppres-
sor p53, which has been shown to interact with Hsp70

and Hsp90 [45,46]. At 25 °C, p53 was efficiently
mono-ubiquitinated by CHIP in the absence of chaper-
ones (Fig. 7A). Neither Hsc70 ⁄ Hdj1 nor Hsp90
enhanced this ubiquitination reaction. At 37 °C,
Hsc70 ⁄ Hdj1 but not Hsp90 stimulated CHIP-mediated
polyubiquitination of p53. Aha1 and p23 had only
minor effects on CHIP-mediated ubiquitination in the
presence of Hsp90 (Fig. 7B). Taken together, as in
the case of luciferase, ubiquitination of the native
Fig. 5. Influence of CHIP on p23 binding to Hsp90. Hsp90 and p23
were incubated in the absence or presence of CHIP, Hop and Aha1
as indicated, and subsequently separated by size-exclusion chroma-
tography on a Superose
TM
12 10 ⁄ 300 column (GE Healthcare, Frei-
burg, Germany), and analyzed by SDS ⁄ PAGE and Coomassie Blue
staining. (A) Representative SDS gels. (B) Quantification of the gels
shown in (A), Fig. S 1 and additional data: the bands representing
p23 were quantified in all lanes. The bar graph shows the amount
of p23 co-eluting with Hsp90 (lanes 4–6) relative to the total
amount of p23 (sum of all lanes).
M. Stankiewicz et al. CHIP preferentially ubiquitinates Hsp70 substrates
FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS 3359
chaperone substrate p53 was more efficient in the pres-
ence of Hsc70 and Hdj1 than in the presence of Hsp90
and its co-chaperones Aha1 or p23.
Discussion
Our study shows that CHIP cooperates with Hsc70
and Hsp90 in a rather passive manner. As CHIP has
no effect on substrate dissociation, ADP dissociation

or ATP association, it does not increase the half-life of
an Hsp70–substrate complex to provide more time for
recruitment of the E2 ubiquitin-conjugating enzyme.
Similarly, CHIP had no influence on the ATPase cycle
of Hsp90 to prolong the ATP-bound state, the ATP-
bound state has a high affinity for substrates. We con-
clude that CHIP to sample available Hsc70–substrate
and Hsp90–substrate complexes in a stochastic process
and thereby occasionally effects ubiquitination. Sub-
strates that are efficiently folded or refolded and there-
fore spend a relatively short time in complex with
Hsc70 or Hsp90 have only a small chance of being
ubiquitinated. In contrast, substrates that cannot be
folded efficiently and consequently cycle on and off the
chaperones continuously, or remain bound to chaper-
one for a prolonged time interval, will eventually be
ubiquitinated by CHIP and targeted for degradation.
Such substrates may be misfolded proteins such as
heat-denatured luciferase, which we have used in
our study, or de novo folding substrates as the cystic
Fig. 6. CHIP-mediated ubiquitination of a denatured substrate is
more efficient in the presence of Hsc70 and Hdj1 than in the pres-
ence of Hsp90. (A–D) Immunoblots of SDS ⁄ PAGE -separated ubiq-
uitination reactions using a luciferase-specific antiserum. (A) Time
course of ubiquitination of heat-denatured firefly luciferase in the
presence of Hsc70 and in the presence and absence of Hdj1. Heat-
denatured firefly luciferase was ubiquitinated in the presence of
50 n
M E1, 1 lM UbcH5c, 1 lM CHIP (except lane 1), 100 lM ubiqu-
itin (except lane 2) and 5 l

M Hsc70, in the absence (lanes 3–8) and
presence (lanes 9–14) of 5 l
M Hdj1 for 1–20 min as indicated. (B)
Comparison of CHIP-dependent poly- and multi-ubiquitination effi-
ciency in the presence of Hsc70 ⁄ Hdj1 and Hsp90. Left panel, ubiq-
uitination of firefly luciferase at various concentrations of Hsc70
(0.2–6 l
M) with 5 lM Hdj1 and various concentrations of Hsp90
(0.2–6 l
M) as indicated. Luciferase was heat-denatured in the pres-
ence of the chaperones, and the ubiquitination mix consisting of
50 n
M E1, 1 lM UbcH5c, 1 lM CHIP and 100 lM ubiquitin was
added. Right panel, CHIP-dependent ubiquitination of heat-dena-
tured luciferase in the presence of 5 l
M Hsc70 plus 5 lM Hdj1
(lanes 9–16) or 5 l
M Hsp90 (lanes 17–24) with wild-type ubiquitin
(lanes 9–12 and 17–20) or the lysine-free ubiquitin variant Ubi-K0, in
which all lysines were replaced by arginines (lanes 13–16 and
21–24) for 10–80 min as indicated. (C) Ubiquitination of firefly lucif-
erase in the presence of various chaperones and co-chaperones.
Lanes 1–12: ubiquitination of luciferase in the presence of 5 l
M
Hsc70 plus 5 lM Hdj1 and the absence (lanes 1–6) or presence
(lanes 7–12) of 5 l
M Bag-1 for 5–120 min as indicated. Lanes 13 to
25: ubiquitination of luciferase in the presence of 5 l
M Hsp90 and
the absence of co-chaperones (lanes 13–17) or the presence of

5 l
M Aha1 (lanes 18–21) or 5 lM p23 (lanes 22–25) for 5–40 min as
indicated. Lane 13 shows the ubiquitination of luciferase in the
absence of CHIP but the presence of Hsp90. (D) CHIP-dependent
ubiquitination of heat-denatured firefly luciferase in the presence of
5 l
M Hsc70 plus 5 lM Hdj1 (lanes 1–5) or 5 lM Hsp90 (lanes 6–16)
and increasing concentrations of Hop (0–23 l
M) for 20 and 120 min
as indicated.
CHIP preferentially ubiquitinates Hsp70 substrates M. Stankiewicz et al.
3360 FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS
fibrosis transmembrane regulator CFTR, a slow-fold-
ing variant (CFTRDF508) of which is known to be
efficiently degraded and has been shown to be ubiquiti-
nated in a CHIP-dependent way [14]. Such a mecha-
nism is also consistent with the phenotype of
CHIP
) ⁄ )
-knockout mice, which accumulate aggregated
proteins [47]. The small amount of proteins that are
ubiquitinated erroneously is the price to be paid for
efficient quality control. Such a mechanism is reminis-
cent of the quality control in the endoplasmic reticu-
lum, where newly synthesized glycoproteins are folded
in the calnexin ⁄ calreticulin cycle [48]. Misfolded glyco-
proteins are bound in turn by the chaperones calnexin
or calreticulin and the folding sensor UDP-glucose-
glycoprotein-glucosyltransferase. Proteins that fold
properly exit this cycle. Glycoproteins that remain in

the cycle for an extended period of time have a high
probability that their N-linked glycans will be trimmed
by a-1,2-mannosidase I, marking the protein for degra-
dation by the ER-associated degradation pathway.
The affinity of dimeric CHIP for dimeric Hsp90
(0.38 lm) was approximately sixfold higher than its
affinity for monomeric Hsc70 (2.3 lm). This result sug-
gests binding of the dimeric CHIP to both C-termini
of the dimeric Hsp90, in agreement with a recent
amide hydrogen exchange study analyzing the interac-
tion of Hsc70 and Hsp90 with CHIP [49]. A K
D
of
2.4 lm was found previously for the interaction of
CHIP with a peptide comprising the ten C-terminal
residues of Hsp90 [1]. This value most likely represents
the K
D
for the initial binding of one TPR domain to a
single EEVD motif of the Hsp90 dimer in a two-step
sequential binding mechanism. K
D
values in the high
nanomolar range have also ben obtained for the inter-
action of other TPR proteins with Hsp90 [27,30].
Therefore, TPR domain proteins compete efficiently
with CHIP for binding to Hsp90, and only a small
amount of Hsp90 is bound to CHIP at equilibrium.
This contrasts with the situation for Hsc70 ⁄ Hsp70,
whose C-termini interact with only the TPR domain

proteins Hop and CHIP [26]. As the concentration of
Hsc70 is greater than the concentrations of Hop and
CHIP together, changes in the CHIP concentration
change the concentration of the Hsc70–CHIP complex,
making the system very sensitive to CHIP concentra-
tions. Despite the lower affinity of CHIP for Hsc70
compared to Hsp90, CHIP is more frequently in com-
plex with Hsc70 in the cell than with Hsp90.
We further demonstrate that ubiquitination of heat-
denatured luciferase is much more efficient in the pres-
ence of Hsc70 and Hdj1 than in the presence of Hsp90.
This observation suggests that misfolded proteins at
least are targeted to the ubiquitin ⁄ proteasomal path-
way through the Hsp70 system rather than through the
Hsp90 system. Such a mechanism might also be true
for bona fide Hsp90 clients once an Hsp90-specific
inhibitor is added. This has been indicated by data for
the glucocorticoid receptor, which was found to
co-localize with Hsp70 and CHIP after addition of gel-
danamycin but not with Hsp90 and FKBP52 [50].
However, if CHIP is over-expressed ectopically or as a
consequence of a pathological process, even Hsp90-
bound clients may be ubiquitinated and degraded [13–
17]. As all Hsp90 clients, which are degraded upon
CHIP over-expression, are also substrates of Hsc70,
the overproduced CHIP may act on the Hsc70–client
complex rather than the Hsp90–client complex. Our
data with p53 support this hypothesis. Therefore, it
Fig. 7. Chip-mediated ubiquitination of the native chaperone sub-
strate p53. (A) Temperature dependence of p53 ubiquitination. p53

was ubiquitinated in the absence and presence of Hsc70 ⁄ Hdj1,
Hsp90 or both at 25 °C (left panel) or 37 °C (right panel). Immuno-
blot of SDS ⁄ PAGE-separated ubiquitination reactions using a
p53-specific antiserum. (B) The Hsp90 co-chaperones Aha1 and
p23 have no influence on CHIP-mediated ubiquitination of p53. p53
was ubiquitinated in the presence of Hsp70 ⁄ Hdj1, Hsp90 and Aha1
and p23 as indicated.
M. Stankiewicz et al. CHIP preferentially ubiquitinates Hsp70 substrates
FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS 3361
remains unclear whether CHIP can selectively ubiquiti-
nate folded Hsp90-associated clients, thereby perform-
ing regulatory functions in the cell. If such a function
of CHIP exists, it seems to be of minor importance, as
the stability of several bona fide Hsp90 substrates is
not affected in CHIP
) ⁄ )
mouse embryonic fibroblasts
[51]. The CHIP
) ⁄ )
mice show phenotypes related to
abnormal protein aggregation [47] rather than break-
down of signaling pathways (compare with FKBP52
knockout mice [52,53]). All these facts speak in favor
of CHIP being an E3 ubiquitin ligase with low sub-
strate specificity that is responsible for the clearance of
hopeless cases of protein folding. However, the role of
CHIP in direct substrate binding and its E4 ligase
function [54,55] remain puzzling.
The native chaperone substrate p53 was only mono-
ubiquitinated by CHIP at 25 °C, and the chaperones

did not enhance this ubiquitination nor stimulate poly-
ubiquitination at this temperature. At 37 °C,
Hsc70 ⁄ Hdj1 but not Hsp90 stimulated CHIP-mediated
polyubiquitination. NMR experiments with p53 core
domain showed that the core domain starts unfolding
at 37 °C and binds concomitantly to Hsp90 [56]. The
Hsc70 ⁄ Hdj1-stimulated polyubiquitination may there-
fore be due to recognition of unfolded regions within
p53 by the chaperone. Therefore, ubiquitination of p53
appears to be very similar to ubiquitination of the
denatured firefly luciferase.
Our study also clarified the previously observed
effect of CHIP on the Hdj1 ⁄ Hdj2-stimulated steady-
state ATPase rate of Hsc70 [14,33]. We show here that
nucleotide exchange of Hsc70 is not affected by CHIP.
In contrast, CHIP decreased the Hdj1-stimulated
c-phosphate cleavage, as demonstrated by single-turn-
over ATPase experiments. CHIP slows down the
transition of Hsc70 from a low-affinity state with high
substrate association and dissociation rates to a
high-affinity state with low substrate dissociation rates.
CHIP therefore counteracts the targeting function of
the J-domain protein. The molecular basis for this
observation could be a reduced association rate for
substrates. It was shown previously that Hsp70 pro-
teins require two signals for highly efficient hydrolysis
of ATP: one signal provided by the J-domain and a
second signal provided by the substrate [57–62]. High
concentrations of some J-domain proteins can provide
both signals by interaction with the substrate binding

pocket as well as the ATPase domain [59,63–65]. As
CHIP reduces the affinity for substrates without affect-
ing the dissociation rate, substrate association is conse-
quently reduced. This in turn reduces the substrate
signal for ATP hydrolysis. It may be advantageous if
substrates do not associate with Hsc70 when CHIP is
already bound. Such a mechanism would prevent ubiq-
uitination of a substrate that has not had the opportu-
nity to refold.
In summary, our results suggest the model shown in
Fig. 8. Proteins in an intermediate folding state after
de novo synthesis at the ribosome or proteins misfolded
under stressful conditions are bound by Hsp70s in an
Hdj-dependent manner and folded ⁄ refolded to the
native state. Proteins that do not fold or that are diffi-
cult to fold are released and rebound by Hsp70s sev-
eral times (black symbols and arrows in Fig. 8). In a
stochastic process, CHIP associates with Hsp70–sub-
strate complexes and recruits the E2 conjugating
enzyme for ubiquitination of the substrate. As Hsp70s
are approximately 10–40 times more abundant than
CHIP, only approximately 1–10% of the Hsp70–
substrate complexes will be bound by CHIP with
possible ubiquitination of the substrate. Efficiently
folding substrates (gray symbols and arrows in Fig. 8)
have only a small chance of being ubiquitinated.
Hsc70–CHIP complexes are less likely to bind misfold-
ed proteins. The likelihood of at least one round of
refolding is thereby increased. This model of the triage
decision allows sufficient time for refolding attempts

by the chaperones, keeping the amount of erroneously
degraded chaperone substrates low. Any increase in
CHIP concentration due to physiological or patho-
physiological processes will increase the clearance rate
for damaged proteins, at an increased cost of degrad-
ing proteins that are still useful.
Experimental procedures
Protein expression and purification
Human CHIP was produced in Escherichia coli and puri-
fied by a combination of cation- and anion-exchange
chromatography as described previously [2]. Human Hdj1
and human Bag-1 were purified as described previously
[37]. Human Hop was purified from over-producing E. coli
strains as described previously [66,67]. All proteins were
quantified as described previously [68] using the Bio-Rad
reagent (Bio-Rad Laboratories, Mu
¨
nchen, Germany).
Human wild-type Hsp90b, Hsc70, Hsp70 and Aha1 were
expressed with an Ulp1 cleavable N-terminal His6–Smt3
tag in E. coli for 5 h at 30 ° C (20 °C for Aha1) and purified
as described previously [69] with some modifications. The
cells were lysed in a French press in 25 mm HEPES ⁄ KOH
pH 7.5, 150 mm KCl, 5 mm MgCl
2
, 5% glycerol, 5 mm
b-mercaptoethanol. Hsp90, Hsc70 and Hsp70 were
further purified on a Resource-Q column (GE Healthcare,
Freiburg, Germany). Hsp90 and Aha1 were further purified
on a Superdex 200 Hiload 16 ⁄ 60 column (GE Healthcare).

CHIP preferentially ubiquitinates Hsp70 substrates M. Stankiewicz et al.
3362 FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS
p23 was expressed as a N-terminal 6His fusion in the same
way, and the histidine tag was removed by digestion with
thrombin protease (Serva, Heidelberg, Germany). The
thrombin and the histidine tag were removed using a
Resource-Q column. UbcH5c was purified as for p23,
except that the histidine tag was not removed. RCMLA
was produced as described previously [70]. Luciferase was
produced as C-terminally His-tagged protein and purified
by metal affinity chromatography. p53 was produced in
E. coli and purified as described previously [46]. Since the
His6-tagged version of ubiquitin was not functional in poly-
ubiquitination assays, wild-type and mutant ubiquitin were
produced in E. coli at 37 °C as untagged proteins, and puri-
fied on SP-Sepharose in 20–500 mm potassium acetate at
pH 4.7 and on a Superdex 75 Hiload 16 ⁄ 60 column
(GE Healthcare) in 25 mm Hepes, pH 7.6, 150 mm KCl,
5mm MgCl
2
, 5% glycerol. The E1 enzyme was purchased
from Calbiochem (Merck Darmstadt, Germany).
Isothermal titration calorimetry
ITC was performed using a VP-ITC calorimeter (Microcal,
Northhampton, MA, USA). Before all titrations, proteins
were dialyzed extensively against ITC buffer (10 mm
Hepes ⁄ KOH (pH 7.4), 150 mm KCl, 5 mm MgCl
2
, 0.5 mm
EDTA). The experiments were performed at 25 °C. A typi-

cal titration consisted of injecting 5–10 lL aliquots of
150 lm CHIP into a solution of 10–15 lm Hsc70, Hsp70 or
Hsc90 at time intervals of 5 min to ensure that the titration
peak returned to baseline. The ITC data were corrected for
the heat of dilution, and analyzed using the software pro-
gram origin version 5.0 provided by the manufacturer.
Substrate binding and dissociation
RCMLA (10 lm) was labeled by incubation with a twofold
molar excess of N-succinimidyl [2,3-
3
H]-propionate (GE
Healthcare, Freiburg, Germany). For substrate dissociation,
<1lm labeled RCMLA was incubated with 10 lm Hsp70
overnight at 16 °C, and then 50 l m unlabeled RCMLA with
or without 50 lm CHIP was added to the pre-formed
complex for the indicated time periods and the complexes
were separated on a Superdex 200 10 ⁄ 300 GL column
(GE Healthcare) and analyzed by scintillation counting. For
substrate binding, Hsp70 (10 lm) was pre-incubated with
50 lm CHIP or buffer control and 30 lm ATP for 20 min
at 37 °C, and RCMLA was added for an additional 1 h.
The binding of p23 to Hsp90 was monitored in the same
way except that the complex was incubated in the presence
of 2 mm ATPcS (Adenosine-5¢-(c-thio)-triphosphate).
Fig. 8. Model for the role of CHIP in the
triage decision. The chaperone systems
encounter various substrates, especially
under heat shock conditions (HS): sub-
strates that can be refolded (gray symbols
and gray arrows) and substrates that cannot

be refolded by the chaperones (black
symbols and black arrows). Substrates that
are efficiently refolded by the Hsp70 system
only require a few cycles of binding and
release by the chaperones and spend a
relatively short time in the chaperone-bound
state. These substrates are only inefficiently
ubiquitinated by CHIP. Substrates that are
not refolded efficiently by the chaperones
(black symbols) are repeatedly bound and
released by the Hsp70 system (black
arrows), and therefore spend a longer time
on the chaperones. Random sampling of
Hsp70 by CHIP entails eventually ubiquitina-
tion and targeting for degradation. If CHIP is
bound to Hsp70, substrate binding to Hsp70
is reduced (light gray arrows).
M. Stankiewicz et al. CHIP preferentially ubiquitinates Hsp70 substrates
FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS 3363
Nucleotide association and dissociation
ATP association and ADP dissociation were performed as
described previously [37] using the fluorescent ADP ⁄ ATP
analogs N
8
-(4-N¢-methylanthraniloylaminobutyl)-8-amino-
adenosine 5¢-di ⁄ triphosphate (MABA-ADP ⁄ MABA-ATP)
[38] and a stopped-flow device (SX-18M, Applied Photo-
physics, Leatherhead, Surrey, UK). To measure ADP disso-
ciation, Hsc70 ⁄ Hsp70 (0.5 lm) were pre-incubated for
30 min with 0.5 lm MABA-ADP in the absence or pres-

ence of 10 lm CHIP in HKM buffer (25 mm Hepes ⁄ KOH
pH 7.6, 50 mm KCL, 5 mm MgCl
2
), and mixed 1 : 1 with a
solution containing 100 lm ATP in the absence or presence
of 0.5 lm Bag-1M and ⁄ or 10 lm CHIP in HKM buffer at
30 °C. To measure ATP association, Hsc70 ⁄ Hsp70 (1 lm)
were pre-incubated in the absence or presence of 10 lm
CHIP and ⁄ or 1 lm Bag-1M in HKM buffer for at least
10 min and then mixed 1 : 1 with 0.5 lm MABA-ATP. Flu-
orescence (excitation 360 nm; cut-off filter at 420 nm) was
measured for 1–100 s at 30 °C, and the time-dependent
changes were fitted using an equation with 1–3 exponential
terms. Data were analyzed using prism 5.0 of graphpad
software ( />Single-turnover ATPase assay
Single-turnover ATPase assays were performed as described
previously [65], isolating complexes of Hsc70 ⁄ Hsp70 with
[a-
32
P]ATP by rapid gel filtration and mixing the pre-
formed complexes with Hdj-1 (1 lm) and CHIP (20 lm).
Hydrolysis products were separated by thin-layer chroma-
tography and quantified on a Fuji fluorophosphoimager
(Fujifilm Europe GmbH, Du
¨
sseldorf, Germany) using
image quant software (Fujifilm). Data were analyzed using
Prism 5.0 of graphpad software.
Steady-state ATPase assay
The steady-state assay was performed as described previ-

ously [71], except the buffer used was 25 mm HEPES pH
7.5, 150 mm KCl, 5 mm MgCl
2
, 5% glycerol, 5 mm
b-mercaptoethanol. The concentration of Hsp90 was
5 lm, that of CHIP was 5–10 times greater than that of
Hsp90, that of Aha1 was three times greater than that of
Hsp90, and that of p23 was ten times greater than that
of Hsp90.
Ubiquitination assay
The assay was performed in 20 mm MOPS pH 7.2, 25 mm
KCl, 25 mm NaCl, 5 mm MgCl
2
,2mm DTT and 5 mm
ATP, with 50 nm E1, 1 lm UbcH5c, 1 lm CHIP, 100 lm
Ubiquitin, 5 lm Hsp90 or 5 lm Hsc70 (or 0.2, 1, 2 and
6 lm for the titration experiment), 5 lm Hdj-1, 5 lm Bag-1,
20 lm Aha1 and p23, 1, 3, 6, 10, 15 and 20 lm Hop, and
1 lm luciferase (from Photinus pyralis) in the indicated
combinations. Luciferase was heat-shocked for 5 min at
43 °C in the presence of the chaperones and the absence of
CHIP, cooled and combined with the ubiquitinating
enzymes. The samples were incubated at 30 °C for the indi-
cated time periods, centrifuged (16 200 g, 10 min, 4 °C),
and the reaction was stopped using SDS loading buffer.
The proteins were separated on SDS ⁄ PAGE and analyzed
by Western blot using a luciferase-specific polyclonal antise-
rum. For ubiquitination of p53, 0.5 lm of the full-length
protein were incubated under the same conditions at the
indicated temperatures for 3 h and analyzed by Western

blot using the antibody Pab 240 (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA).
Acknowledgements
We like to thank B. Bukau for support and discus-
sions. This work was supported by grants from the
Deutsche Forschungsgemeinschaft (SFB638) and the
BioQuant Promotionskolleg of Baden-Wu
¨
rttemberg.
References
1 Zhang M, Windheim M, Roe SM, Peggie M, Cohen P,
Prodromou C & Pearl LH (2005) Chaperoned ubiquity-
lation – crystal structures of the CHIP U box E3 ubiqu-
itin ligase and a CHIP–Ubc13–Uev1a complex. Mol
Cell 20, 525–538.
2 Nikolay R, Wiederkehr T, Rist W, Kramer G, Mayer
MP & Bukau B (2004) Dimerization of the human E3
ligase CHIP via a coiled-coil domain is essential for its
activity. J Biol Chem 279, 2673–2678.
3 Cyr DM, Hohfeld J & Patterson C (2002) Protein qual-
ity control: U-box-containing E3 ubiquitin ligases join
the fold. Trends Biochem Sci 27, 368–375.
4Ho
¨
hfeld J, Cyr DM & Patterson C (2001) From the
cradle to the grave: molecular chaperones that may
choose between folding and degradation. EMBO Rep 2,
885–890.
5 Wiederkehr T, Bukau B & Buchberger A (2002) Protein
turnover: a CHIP programmed for proteolysis. Curr

Biol 12, R26–R28.
6 Bukau B & Horwich AL (1998) The Hsp70 and Hsp60
chaperone machines. Cell 92, 351–366.
7 Liberek K, Lewandowska A & Zietkiewicz S (2008)
Chaperones in control of protein disaggregation.
EMBO J 27, 328–335.
8 Mayer MP & Bukau B (2005) Hsp70 chaperones: cellu-
lar functions and molecular mechanism. Cell Mol Life
Sci 62, 670–684.
9 Jakob U, Lilie H, Meyer I & Buchner J (1995) Tran-
sient interaction of Hsp90 with early unfolding
CHIP preferentially ubiquitinates Hsp70 substrates M. Stankiewicz et al.
3364 FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS
intermediates of citrate synthase. Implications for heat
shock in vivo. J Biol Chem 270, 7288–7294.
10 Wegele H, Muller L & Buchner J (2004) Hsp70 and
Hsp90 – a relay team for protein folding. Rev Physiol
Biochem Pharmacol 151, 1–44.
11 Picard D (2002) Heat-shock protein 90, a chaperone for
folding and regulation. Cell Mol Life Sci 59, 1640–1648.
12 Pearl LH, Prodromou C & Workman P (2008) The
Hsp90 molecular chaperone: an open and shut case for
treatment. Biochem J 410, 439–453.
13 Connell P, Ballinger CA, Jiang J, Wu Y, Thompson LJ,
Hohfeld J & Patterson C (2001) The co-chaperone
CHIP regulates protein triage decisions mediated by
heat-shock proteins. Nat Cell Biol 3, 93–96.
14 Meacham GC, Patterson C, Zhang W, Younger JM &
Cyr DM (2001) The Hsc70 co-chaperone CHIP targets
immature CFTR for proteasomal degradation. Nat Cell

Biol 3, 100–105.
15 Zhou P, Fernandes N, Dodge IL, Reddi AL, Rao N,
Safran H, DiPetrillo TA, Wazer DE, Band V & Band
H (2003) ErbB2 degradation mediated by the co-chaper-
one protein CHIP. J Biol Chem 278, 13829–13837.
16 Adachi H, Waza M, Tokui K, Katsuno M, Minamiy-
ama M, Tanaka F, Doyu M & Sobue G (2007) CHIP
overexpression reduces mutant androgen receptor pro-
tein and ameliorates phenotypes of the spinal and bul-
bar muscular atrophy transgenic mouse model.
J Neurosci 27, 5115–5126.
17 Kamynina E, Kauppinen K, Duan F, Muakkassa N &
Manor D (2007) Regulation of proto-oncogenic Dbl by
chaperone-controlled, ubiquitin-mediated degradation.
Mol Cell Biol 27, 1809–1822.
18 Rosser MF, Washburn E, Muchowski PJ, Patterson C
& Cyr DM (2007) Chaperone functions of the E3
ubiquitin ligase CHIP. J Biol Chem 282, 22267–22277.
19 Li RF, Zhang F, Lu YJ & Sui SF (2005) Specific inter-
action between Smad1 and CHIP: a surface plasmon
resonance study. Colloids Surf B Biointerfaces 40, 133–
136.
20 Xin H, Xu X, Li L, Ning H, Rong Y, Shang Y,
Wang Y, Fu XY & Chang Z (2005) CHIP controls
the sensitivity of transforming growth factor-b signal-
ing by modulating the basal level of Smad3 through
ubiquitin-mediated degradation. J Biol Chem 280,
20842–20850.
21 Xu W, Marcu M, Yuan X, Mimnaugh E, Patterson C
& Neckers L (2002) Chaperone-dependent E3 ubiquitin

ligase CHIP mediates a degradative pathway for c-
ErbB2 ⁄ Neu. Proc Natl Acad Sci USA 99, 12847–12852.
22 Younger JM, Ren HY, Chen L, Fan CY, Fields A,
Patterson C & Cyr DM (2004) A foldable CFTRDF508
biogenic intermediate accumulates upon inhibition of
the Hsc70–CHIP E3 ubiquitin ligase. J Cell Biol 167,
1075–1085.
23 Tateishi Y, Kawabe Y, Chiba T, Murata S, Ichikawa
K, Murayama A, Tanaka K, Baba T, Kato S & Yana-
gisawa J (2004) Ligand-dependent switching of ubiqu-
itin–proteasome pathways for estrogen receptor. EMBO
J 23, 4813–4823.
24 Smith DF (1993) Dynamics of heat shock protein 90–
progesterone receptor binding and the disactivation
loop model for steroid receptor complexes. Mol Endo-
crinol 7, 1418–1429.
25 Pratt WB & Toft DO (1997) Steroid receptor interac-
tions with heat shock protein and immunophilin chaper-
ones. Endocr Rev 18, 306–360.
26 Smith DF (2004) Tetratricopeptide repeat cochaperones
in steroid receptor complexes.
Cell Stress Chaperones 9,
109–121.
27 Wegele H, Haslbeck M, Reinstein J & Buchner J (2003)
Sti1 is a novel activator of the Ssa proteins. J Biol
Chem 278, 25970–25976.
27a Lai BT, Chin NW, Stanek AE, Keh W & Lanks KW
(1984) Quantitation and intracellular localization of the
85K heat shock protein by using monoclonal and poly-
clonal antibodies. Mol Cell Biol 4, 2802–2810.

28 Gillen CM & Forbush B III (1999) Functional interac-
tion of the K–Cl cotransporter (KCC1) with the Na–K–
Cl cotransporter in HEK-293 cells. Am J Physiol 276,
C328–C336.
29 Fumarola C, La Monica S, Alfieri RR, Borra E & Gui-
dotti GG (2005) Cell size reduction induced by inhibi-
tion of the mTOR ⁄ S6K-signaling pathway protects
Jurkat cells from apoptosis. Cell Death Differ 12, 1344–
1357.
30 Pirkl F & Buchner J (2001) Functional analysis of the
Hsp90-associated human peptidyl prolyl cis ⁄ trans
isomerases FKBP51, FKBP52 and Cyp40. J Mol Biol
308, 795–806.
31 Prodromou C, Siligardi G, O’Brien R, Woolfson DN,
Regan L, Panaretou B, Ladbury JE, Piper PW & Pearl
LH (1999) Regulation of Hsp90 ATPase activity by
tetratricopeptide repeat (TPR)-domain co-chaperones.
EMBO J 18, 754–762.
32 Palleros DR, Welch WJ & Fink AL (1991) Interaction
of hsp70 with unfolded proteins: effects of temperature
and nucleotides on the kinetics of binding. Proc Natl
Acad Sci USA 8, 5719–5723.
33 Ballinger CA, Connell P, Wu Y, Hu Z, Thompson LJ,
Yin LY & Patterson C (1999) Identification of CHIP,
a novel tetratricopeptide repeat-containing protein that
interacts with heat shock proteins and negatively regu-
lates chaperone functions. Mol Cell Biol 19, 4535–
4545.
34 Ha J-H & McKay DB (1994) ATPase kinetics of recom-
binant bovine 70 kDa heat shock cognate protein and

its amino-terminal ATPase domain. Biochemistry 33,
14625–14635.
M. Stankiewicz et al. CHIP preferentially ubiquitinates Hsp70 substrates
FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS 3365
35 Ha J-H, Johnson ER, McKay DB, Sousa MC, Takeda
S & Wilbanks SM (1999) Structure and mechanism of
Hsp70 proteins. In Molecular Chaperones and Folding
Catalysts: Regulation, Cellular Function and Mechanism
(Bukau B ed), pp 573–607. Harwood Academic Publish-
ers, Amsterdam.
36 Brehmer D, Ru
¨
diger S, Ga
¨
ssler CS, Klostermeier D,
Packschies L, Reinstein J, Mayer MP & Bukau B
(2001) Tuning of chaperone activity of Hsp70 proteins
by modulation of nucleotide exchange. Nat Struct Biol
8, 427–432.
37 Ga
¨
ssler CS, Wiederkehr T, Brehmer D, Bukau B &
Mayer MP (2001) Bag-1M accelerates nucleotide release
for human Hsc70 and Hsp70 and can act concentra-
tion-dependent as positive and negative cofactor. J Biol
Chem 276, 32538–32544.
38 Theyssen H, Schuster HP, Packschies L, Bukau B &
Reinstein J (1996) The second step of ATP binding to
DnaK induces peptide release. J Mol Biol 263, 657–670.
39 Ho

¨
hfeld J & Jentsch S (1997) GrpE-like regulation of
the Hsc70 chaperone by the anti-apoptotic protein
BAG-1. EMBO J 16, 6209–6216.
40 Demand J, Alberti S, Patterson C & Hohfeld J (2001)
Cooperation of a ubiquitin domain protein and an E3
ubiquitin ligase during chaperone ⁄ proteasome coupling.
Curr Biol 11, 1569–1577.
41 Panaretou B, Siligardi G, Meyer P, Maloney A, Sulli-
van JK, Singh S, Millson SH, Clarke PA, Naaby-Han-
sen S, Stein R et al. (2002) Activation of the ATPase
activity of hsp90 by the stress-regulated cochaperone
aha1. Mol Cell 10, 1307–1318.
42 McLaughlin SH, Smith HW & Jackson SE (2002) Stim-
ulation of the weak ATPase activity of human hsp90 by
a client protein. J Mol Biol 315, 787–798.
43 Hernandez MP, Sullivan WP & Toft DO (2002) The
assembly and intermolecular properties of the hsp70–
Hop–hsp90 molecular chaperone complex. J Biol Chem
2, 38294–38304.
44 Young JC & Hartl FU (2000) Polypeptide release by
Hsp90 involves ATP hydrolysis and is enhanced by the
co-chaperone p23. EMBO J 19, 5930–5940.
45 Muller L, Schaupp A, Walerych D, Wegele H & Buch-
ner J (2004) Hsp90 regulates the activity of wild type
p53 under physiological and elevated temperatures.
J Biol Chem 279, 48846–48854.
46 King FW, Wawrzynow A, Hohfeld J & Zylicz M (2001)
Co-chaperones Bag-1, Hop and Hsp40 regulate Hsc70
and Hsp90 interactions with wild-type or mutant p53.

EMBO J 20, 6297–6305.
47 Min JN, Whaley RA, Sharpless NE, Lockyer P, Port-
bury AL & Patterson C (2008) CHIP deficiency
decreases longevity, with accelerated aging phenotypes
accompanied by altered protein quality control. Mol
Cell Biol 28, 4018–4025.
48 Ellgaard L & Helenius A (2003) Quality control in the
endoplasmic reticulum. Nat Rev Mol Cell Biol 4, 181–
191.
49 Graf C, Stankiewicz M, Nikolay R & Mayer MP (2010)
Insights into the conformational dynamics of the E3
ubiquitin ligase CHIP in complex with chaperones and
E2 enzymes. Biochemistry 49, 2121–2129.
50 Galigniana MD, Harrell JM, Housley PR, Patterson C,
Fisher SK & Pratt WB (2004) Retrograde transport of
the glucocorticoid receptor in neurites requires dynamic
assembly of complexes with the protein chaperone
hsp90 and is linked to the CHIP component of the
machinery for proteasomal degradation. Brain Res Mol
Brain Res 123, 27–36.
51 Morishima Y, Wang AM, Yu Z, Pratt WB, Osawa Y &
Lieberman AP (2008) CHIP deletion reveals functional
redundancy of E3 ligases in promoting degradation of
both signaling proteins and expanded glutamine pro-
teins. Hum Mol Genet 17, 3942–3952.
52 Yang Z, Wolf IM, Chen H, Periyasamy S, Chen Z,
Yong W, Shi S, Zhao W, Xu J, Srivastava A et al.
(2006) FK506-binding protein 52 is essential to uterine
reproductive physiology controlled by the progesterone
receptor A isoform. Mol Endocrinol 20, 2682–2694.

53 Cheung-Flynn J, Prapapanich V, Cox MB, Riggs DL,
Suarez-Quian C & Smith DF (2005) Physiological role
for the cochaperone FKBP52 in androgen receptor sig-
naling. Mol Endocrinol 19, 1654–1666.
54 Hoppe T, Cassata G, Barral JM, Springer W, Hutaga-
lung AH, Epstein HF & Baumeister R (2004) Regula-
tion of the myosin-directed chaperone UNC-45 by a
novel E3 ⁄ E4-multiubiquitylation complex in C. elegans.
Cell 118, 337–349.
55 Imai Y, Soda M, Hatakeyama S, Akagi T, Hashikawa
T, Nakayama KI & Takahashi R (2002) CHIP is asso-
ciated with parkin, a gene responsible for familial Par-
kinson’s disease, and enhances its ubiquitin ligase
activity. Mol Cell 10, 55–67.
56 Ru
¨
diger S, Freund SM, Veprintsev DB & Fersht AR
(2002) CRINEPT-TROSY NMR reveals p53 core
domain bound in an unfolded form to the chaperone
Hsp90. Proc Natl Acad Sci USA 99, 11085–11090.
57 Karzai AW & McMacken R (1996) A bipartite signal-
ing mechanism involved in DnaJ-mediated activation of
the Escherichia coli DnaK protein. J Biol Chem 271,
11236–11246.
58 Barouch W, Prasad K, Greene L & Eisenberg E (1997)
Auxilin-induced interaction of the molecular chaperone
Hsc70 with clathrin baskets. Biochemistry 36, 4303–
4308.
59 Laufen T, Mayer MP, Beisel C, Klostermeier D, Mogk
A, Reinstein J & Bukau B (1999) Mechanism of regula-

tion of hsp70 chaperones by DnaJ cochaperones. Proc
Natl Acad Sci USA 96, 5452–5457.
CHIP preferentially ubiquitinates Hsp70 substrates M. Stankiewicz et al.
3366 FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS
60 Silberg JJ, Tapley TL, Hoff KG & Vickery LE (2004)
Regulation of the HscA ATPase reaction cycle by the
co-chaperone HscB and the iron–sulfur cluster assembly
protein IscU. J Biol Chem 279, 53924–53931.
61 Han W & Christen P (2003) Mechanism of the targeting
action of DnaJ in the DnaK molecular chaperone sys-
tem. J Biol Chem 278 , 19038–19043.
62 Siegenthaler RK & Christen P (2006) Tuning of DnaK
chaperone action by nonnative protein sensor DnaJ and
thermosensor GrpE. J Biol Chem 281, 34448–34456.
63 Suh W-C, Lu CZ & Gross CA (1999) Structural fea-
tures required for the interaction of the Hsp70 molecu-
lar chaperone DnaK with its cochaperone DnaJ. J Biol
Chem 274, 30534–30539.
64 Misselwitz B, Staeck O & Rapoport TA (1998)
J proteins catalytically activate Hsp70 molecules to
trap a wide range of peptide sequences. Mol Cell 2,
593–603.
65 Mayer MP, Laufen T, Paal K, McCarty JS & Bukau B
(1999) Investigation of the interaction between DnaK
and DnaJ by surface plasmon resonance spectroscopy.
J Mol Biol 289, 1131–1144.
66 Buchner J, Weikl T, Bugl H, Pirkl F & Bose S (1998)
Purification of Hsp90 partner proteins Hop ⁄ p60, p23,
and FKBP52. Methods Enzymol 290, 418–429.
67 Buchner J, Bose S, Mayr C & Jakob U (1998) Purifica-

tion and characterization of prokaryotic and eukaryotic
Hsp90. Methods Enzymol 290, 409–418.
68 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein
utilizing the principle of protein–dye binding. Anal
Biochem 72, 248–254.
69 Andreasson C, Fiaux J, Rampelt H, Mayer MP & Buk-
au B (2008) Hsp110 is a nucleotide-activated exchange
factor for Hsp70. J Biol Chem 283, 8877–8884.
70 Bosl B, Grimminger V & Walter S (2005) Substrate
binding to the molecular chaperone Hsp104 and its
regulation by nucleotides. J Biol Chem 280, 38170–
38176.
71 Ali JA, Jackson AP, Howells AJ & Maxwell A (1993)
The 43-kilodalton N-terminal fragment of the DNA
gyrase B protein hydrolyzes ATP and binds coumarin
drugs. Biochemistry 32, 2717–2724.
72 Longshaw VM, Dirr HW, Blatch GL & Lassle M
(2000) The in vitro phosphorylation of the co-chaperone
mSTI1 by cell cycle kinases substantiates a predicted
casein kinase II–p34cdc2–NLS (CcN) motif. Biol Chem
381, 1133–1138.
73 Daniel S, Bradley G, Longshaw VM, Soti C, Csermely
P & Blatch GL (2008) Nuclear translocation of the
phosphoprotein Hop (Hsp70 ⁄ Hsp90 organizing protein)
occurs under heat shock, and its proposed nuclear local-
ization signal is involved in Hsp90 binding. Biochim
Biophys Acta 1783, 1003–1014.
Supporting information
The following supplementary material is available:

Fig. S1. Gel filtration analysis of Hsp90, CHIP, p23
and combinations thereof.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
M. Stankiewicz et al. CHIP preferentially ubiquitinates Hsp70 substrates
FEBS Journal 277 (2010) 3353–3367 ª 2010 The Authors Journal compilation ª 2010 FEBS 3367