Molecular Biology of the Cell
Vol. 16, 1165–1177, March 2005
J-Domain Protein CDJ2 and HSP70B Are a Plastidic
Chaperone Pair That Interacts with Vesicle-Inducing
Protein in Plastids 1
Cuimin Liu,* Felix Willmund,* Julian P. Whitelegge,†Susan Hawat,‡
Bettina Knapp,* Mukesh Lodha,* and Michael Schroda*
*Plant Biochemistry, Institute of Biology II, University of Freiburg, D-79104 Freiburg, Germany;†Department
of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095; and‡Institute
of General Botany, University of Jena, 07743 Jena, Germany
Submitted August 25, 2004; Revised November 18, 2004; Accepted December 8, 2004
Monitoring Editor: Reid Gilmore
J-domain cochaperones confer functional specificity to their heat shock protein (HSP)70 partner by recruiting it to specific
substrate proteins. To gain insight into the functions of plastidic HSP70s, we searched in Chlamydomonas databases for
expressed sequence tags that potentially encode chloroplast-targeted J-domain cochaperones. Two such cDNAs were
found: the encoded J-domain proteins were named chloroplast DnaJ homolog 1 and 2 (CDJ1 and CDJ2). CDJ2 was shown
to interact with a ?28-kDa protein that by mass spectrometry was identified as the vesicle-inducing protein in plastids 1
(VIPP1). In fractionation experiments, CDJ2 was detected almost exclusively in the stroma, whereas VIPP1 was found in
low-density membranes, thylakoids, and in the stroma. Coimmunoprecipitation and mass spectrometry analyses identi-
fied stromal HSP70B as the major protein interacting with soluble VIPP1, and, as confirmed by cross-linking data, as
chaperone partner of CDJ2. In blue native-PAGE of soluble cell extracts, CDJ2 and VIPP1 comigrated in complexes of
? ?669, ?150, and perhaps ?300 kDa. Our data suggest that CDJ2, presumably via coiled-coil interactions, binds to VIPP1
and presents it to HSP70B in the ATP state. Our findings and the previously reported requirement of VIPP1 for the
biogenesis of thylakoid membranes point to a role for the HSP70B/CDJ2 chaperone pair in this process.
Chaperones of the heat shock protein (Hsp)70 family belong
to the most conserved proteins known. Except for some
Archaea, Hsp70s are found in all known organisms and are
present in every compartment of the eukaryotic cell (Bukau
and Horwich, 1998). Principally, Hsp70s consist of an N-
terminal ATPase domain and a C-terminal substrate-bind-
ing domain. ATP hydrolysis at the ATPase domain regulates
substrate binding and release. Substrate proteins recognized
by Hsp70 expose hydrophobic regions, a characteristic fea-
ture not only of nonnative proteins, but also of native Hsp70
substrates. Binding of Hsp70 to hydrophobic regions pre-
vents the formation of aggregates. In addition, the intrinsic
secondary amide peptide bond cis-trans isomerase activity
recently detected for DnaK (the Hsp70 of Escherichia coli)
may introduce conformational changes to bound substrates
that eventually allow nonnative proteins to reconvert to the
native state (Schiene-Fischer et al., 2002). Thus, Hsp70s play
a major role in the folding of nascent chains and in the
renaturation of nonnative proteins that have accumulated
during stress situations such as heat shock (Frydman, 2001).
However, they also are involved in many highly specialized
functions such as the regulation of the general stress re-
sponse (Tomoyasu et al., 1998), the uncoating of clathrin-
coated vesicles (Ungewickell et al., 1995), or the translocation
of proteins across membranes (Kang et al., 1990).
Specificity of Hsp70 function is mediated largely by its
cochaperones, of which the J-domain cochaperones repre-
sent an important class. J-domain cochaperones contain a
highly conserved J-domain that is responsible for the inter-
action with Hsp70. In addition, these cochaperones contain
domains typical for protein–protein interactions, such as
zinc finger or coiled-coil domains, by which specific sub-
strates are bound (Cyr et al., 1994; Szabo et al., 1996; Miernyk,
2001). These substrates are then presented to the substrate
binding domain of a specific Hsp70 chaperone. One Hsp70
may be recruited by several different J-domain cochaperones
to fulfill a variety of specific tasks. In the Arabidopsis thaliana
genome, for example, at least 89 genes encoding J-domain
proteins (Miernyk, 2001) cooperate with only 12 Hsp70s
(Sung et al., 2001).
Three Hsp70 systems have been identified in the chloro-
plast (Schroda, 2004). Two of these, Com70 and Hsp70 IAP,
seem to be involved in the import of precursor proteins into
the chloroplast (Schnell et al., 1994; Kourtz and Ko, 1997).
The third plastidic Hsp70 system is located to the stroma.
Stromal Hsp70 has been shown to cooperate with Cpn60 in
the folding of proteins newly imported into the chloroplast
(Maduen ˜o et al., 1993; Tsugeki and Nishimura, 1993; Bonk et
al., 1996) and indirect evidence suggested that it also is
involved in the refolding of denatured proteins (Schroda et
Also chloroplast-specific functions can be attributed to
stromal Hsp70. In Chlamydomonas, stromal HSP70B was
This article was published online ahead of print in MBC in Press
on January 5, 2005.
Address correspondence to: Michael Schroda (michael.schroda@biologie.
© 2005 by The American Society for Cell Biology1165
shown to increase the cell’s ability to cope with photoinhi-
bition, possibly by either protecting photosystem II from
irreversible photodamage or by stabilizing photodamaged
photosystem II for a coordinated exchange of damaged D1
protein for de novo-synthesized D1 (Schroda et al., 1999,
2001a). Other specialized functions of stromal Hsp70 were
deduced from functions attributed to plastidic DnaJ-like
proteins. The maize bsd2 mutant impaired in Rubisco assem-
bly was shown to lack a protein exhibiting high sequence
similarity to the zinc finger domain of DnaJ proteins (Brut-
nell et al., 1999). Bsd2 was hypothesized to act together with
ATJ11, which consists essentially of a J-domain to recruit a
stromal Hsp70 for the folding of Rubisco subunits (Orme et
al., 2001). An Arabidopsis mutant impaired in chloroplast
division was shown to be defective in the ARC6 J-domain–
like protein. ARC6 spans the inner envelope membrane and
exposes its J-domain to the stroma, where it may recruit a
stromal Hsp70 for a function in chloroplast division (Vitha et
Evidently, insight into the functions of chloroplast Hsp70s
may be gained by the functional characterization of chloro-
plast-localized J-domain proteins. For this purpose, we have
identified expressed sequence tags (ESTs) from Chlamydomo-
nas databases that encode proteins harboring both a putative
chloroplast transit peptide and a J-domain. In this study, we
show that the chloroplast-targeted J-domain protein CDJ2
interacts with the vesicle-inducing protein in plastids 1
(VIPP1) and that both CDJ2 and VIPP1 interact with stromal
HSP70B. VIPP1 (or IM30) was first described as a 30-kDa
protein associated with the inner envelope and the thylakoid
membranes of pea chloroplasts (Li et al., 1994). From this
unusual localization, the authors proposed that VIPP1 might
be involved in lipid transfer from the inner envelope to the
thylakoids, perhaps by vesicle traffic (Li et al., 1994). Consis-
tent with this hypothesis is the phenotype of the Arabidopsis
hcf155 mutant, which expresses the VIPP1 gene at signifi-
cantly lower levels, and, as a result, exhibits dramatic defects
in the structure of its thylakoid membranes (Kroll et al.,
2001). In addition, vesicle budding from the inner envelope
was found to be abolished in hcf155. Also, in Synechocystis
cells that expressed VIPP1 at low levels, distorted thylakoids
were observed (Westphal et al., 2001). The data presented
here suggest that the stromal HSP70B/CDJ2 chaperone pair
might play a role in the functional cycle of VIPP1 required
for the biogenesis/maintenance of thylakoid membranes.
MATERIALS AND METHODS
Strains and Culture Conditions
Chlamydomonas reinhardtii strains were grown mixotrophically in TAP me-
dium (Harris, 1989) on a rotatory shaker at 25°C at ?30 ?E m?2s?1. For
chloroplast isolation, cells were grown in TAP medium supplemented with
Heat Shock and Dark-to-Light Shift Kinetics, RNA and
Protein Extractions, RNA Gels, and Hybridizations
For heat shock kinetics, Chlamydomonas cc124 cells were grown in 160 ml of
TAP medium to a density of 6 ? 106cells/ml, harvested by centrifugation,
and resuspended in 100 ml of TAP medium prewarmed to 40°C. For dark-
to-light shift, Chlamydomonas cc124 cells were grown to a density of 6 ? 106
cells/ml, incubated in the dark for 16 h, and transferred to dim light (?30 ?E
m?2s?1). Samples were taken at different time points and cooled by adding
ice. Cells were harvested by centrifugation and resuspended in 600 ?l of TAP
medium, of which 100 ?l for protein analyses were centrifuged; resuspended
in 40 ?l of 0.1 M NaCO3, 0.1 M dithiothreitol; and solubilized by adding 55 ?l
of 5%SDS, 30% sucrose. For RNA extraction, 500 ?l of lysis buffer (100 mM
Tris-HCl, pH 8.0, 600 mM NaCl, 4% SDS, and 10 mM EDTA) was added to the
remaining 500 ?l of cell suspension, followed by incubation at 65°C for 10
min. SDS was precipitated by adding 132 ?l of 2 M KCl, incubation on ice for
15 min, and centrifugation. Remaining proteins were extracted once with
phenol/chloroform and once with chloroform. After addition of 0.3 volumes
of 8 M LiCl and incubation at 4°C overnight, RNA was pelleted by centrifu-
gation. RNA was resuspended in diethylpyrocarbonate (DEPC)-treated wa-
ter, precipitated with ethanol, and resuspended in DEPC-treated water. RNA
(10 ?g) per lane was separated on 1.2% formaldehyde agarose gels (Sambrook
et al., 1989), incubated for 30 min with 50 mM NaOH, 10 mM NaCl and for 10
min with 100 mM Tris-HCl, pH 7.5, and blotted to Hybond-N membranes
(Amersham Biosciences, Freiburg, Germany) by capillary transfer with 25
mM phosphate buffer, pH 6.5. After baking for 2 h at 80°C, membranes were
hybridized with DNA probes prepared by the random priming technique
(Feinberg and Vogelstein, 1983) by using [?-32P]dCTP (Amersham Bio-
sciences). Hybridization and quantitation was done as described previously
(Schroda et al., 1999). Probes used were a 2-kb NheI-AatII fragment containing
the HSP70B coding region, a 1.9-kb SpeI-XhoI fragment containing the CDJ1
cDNA (AV626034), a 1.35-kb polymerase chain reaction (PCR) product con-
taining the VIPP1 coding region and 3? untranslated region, and a 1-kb cDNA
of c?lp2 (Von Kampen et al., 1994).
Polyacrylamide Electrophoreses and Gel Blot Analyses
Blue native (BN)-PAGE for soluble proteins was carried out according to a
published protocol (Scha ¨gger et al., 1994). The native high-molecular-weight
marker (66–669 kDa) was purchased from Amersham Biosciences. SDS-
PAGE was performed as described previously (Laemmli, 1970). For heat
shock kinetics, proteins were loaded on the basis of equal chlorophyll con-
centrations. For fractionation experiments, 1 volume of 2? Laemmli sample
buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% ?-mercapto-
ethanol, and 0.005% bromphenol blue) was added to the samples, and protein
concentrations were determined by amido black (Popov et al., 1975).
Proteins in gels were stained with silver nitrate or transferred to nitrocel-
lulose membranes (Hybond-ECL; Amersham Biosciences) by semidry blot-
ting by using a discontinuous transfer system. Blocking and immunodecora-
tions were performed in phosphate-buffered saline (Sambrook et al., 1989)
containing 3% nonfat dry milk, immunodetection was done by enhanced
chemiluminescence (Amersham Biosciences). Antisera described previously
were against HSP70B (Schroda et al., 1999), CGE1 (Schroda et al., 2001b),
mitochondrial carboanhydrase (Eriksson et al., 1996), and Cytf (Pierre and
Chlamydomonas cc124 was grown to a density of ?5 ? 106cells/ml, har-
vested by centrifugation, and resuspended in lysis buffer (20 mM HEPES, pH
7.2, 10 mM KCl, 1 mM MgCl2, 154 mM NaCl, 10% glycerol, and 0.25?
protease inhibitor cocktail; Roche Diagnostics, Mannheim, Germany). For the
experiment in Figure 6, cells were split in two parts, to one-half carbonyl
cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at a final concentration
of 20 ?M was added, and then both halves were incubated on a shaker for 10
min at 25°C. To the FCCP-treated cells apyrase was added at a concentration
of 0.3 U/ml; to nontreated cells Mg-ATP was added to a final concentration
of 2.5 mM, and both halves were incubated on ice for 10 min before sonica-
tion. For all experiments, cells were sonicated on ice for 90 s. Lysates were
loaded onto sucrose cushions (20 mM HEPES-KOH, pH 7.2, 0.6 M sucrose)
and centrifuged in a TI50 rotor for 30 min at 152,000 ? g and 4°C. For the
experiments in Figures 5 and 6, supernatants were supplied with Triton X-100
to a final concentration of 0.5%. Protein A-Sepharose beads with coupled
antibodies were equilibrated in lysis buffer and incubated with the cell lysates
under agitation for 1 h at 4°C. Beads were washed four times with lysis buffer
(containing 0.1% Triton X-100) and once with 10 mM Tris-HCl, pH 7.5, and
proteins were eluted by boiling 45 s in 1? Laemmli sample buffer (Figure 3)
or by shaking 30 min at 25°C with 1? Laemmli sample buffer lacking
?-mercaptoethanol (Figures 5 and 6).
All fractionations were carried out with cw15 strain CF185 (Schroda et al.,
1999). Cells were grown to ?5 ? 106cells/ml, harvested by centrifugation,
and resuspended in 10 mM Tris-HCl, pH 7.6, 0.25? protease inhibitor cock-
tail. Cells were split into two parts and ruptured by either sonication on ice or
four cycles of freeze-thawing. Broken cells were centrifuged in a TLA 100.2
rotor for 1 h at 355,000 ? g and 4°C. The supernatant was regarded as soluble
proteins and the pellet after resuspension in the initial volume of Tris-HCl
buffer as membranes. Isolation of chloroplasts and fractionation into stroma,
thylakoids, and low-density membranes was done as described previously
(Zerges and Rochaix, 1998). Mitochondria were isolated following a pub-
lished protocol (Eriksson et al., 1995).
Mass Spectrometry Analyses
Proteins in gels were visualized with Coomassie or silver staining and bands
were excised and treated with trypsin before extraction of peptides for anal-
ysis by microliquid chromatography tandem mass spectrometry (?LC-
MSMS) (Shevchenko et al., 1996). Samples were analyzed by ?LC-MSMS with
data-dependent acquisition (LCQ-DECA; Thermo Finnigan, San Jose, CA)
after dissolution in 5 ?l of 70% acetic acid (vol/vol). A reverse-phase column
C. Liu et al.
Molecular Biology of the Cell1166
(200 ?m ? 10 cm; PLRP/S 5 ?m, 300 Å; Michrom Biosciences, San Jose, CA)
was equilibrated for 10 min at 1.5 ?l/min with 95% A, 5% B (A, 0.1% formic
acid in water; B, 0.1% formic acid in acetonitrile) before sample injection. A
linear gradient was initiated 10 min after sample injection ramping to 60% A,
40% B after 50 min and 20% A, 80% B after 65 min. Column eluent was
directed to a coated glass electrospray emitter (TaperTip, TT150-50-50-CE-5;
New Objective, Woburn, MA) at 3.3 kV for ionization without nebulizer gas.
The mass spectrometer was operated in “triple-play” mode with a survey
scan (400–1500 m/z), data-dependent zoom scan, and MSMS with exclusion of
singly charged ions (Whitelegge, 2003). Individual sequencing experiments
were matched to a custom Chlamydomonas sequence database downloaded
from JGI (http://www.jgi.doe.gov/) by using Sequest software (Thermo
Finnigan). The search was run under the “no enzyme” mode to identify
nontryptic peptides. The results of Sequest searches were carefully scruti-
nized. MSMS spectra of doubly charged ions with cross-correlation scores
(Xcorr) ?2.8, and triply charged ions with scores ?3.2 were examined man-
ually. Some noisy spectra were discarded despite high Xcorr scores. Nontryp-
tic peptide returns were retained only if the data looked of especially high
signal to noise.
Cloning, Expression, and Purification of CDJ2, CD?2, and
The coding region of CDJ2 was amplified by PCR from cDNA clone AV387908
with primers 5?-AAGGATCCATGGCAGCGAAGAACTTCTACGAC-3? and
5?-CGTGGGTAACCTAGCTTATTGAGCTTCTTCTTGAGC-3?. The ?1-kb PCR
product was digested with BamHI and BstEII and cloned into BamHI-BstEII–
digested pCB785 (a pQE-9 derivative containing the HSP70B coding region
cloned into BamHI and BstEII restriction sites), giving pMS254. The coding
region of CDJ2 excluding the J-domain was amplified by PCR with primers
5?-GCTGCCCATGGGCTACGCCGGCGGGAGGA-3? and 5?-CGTGGGTA-
ACCTAGCTTATTGAGCTTCTTCTTGAGC-3?; digested with NcoI and BstEII;
and after a subcloning step, ligated into pMS254, yielding pMS269. pMS254
and 269 were expressed in E. coli M15 (QIAGEN, Hilden, Germany) and
purified by nickel-nitrilotriacetic acid agarose (Ni-NTA) according to the
manufacturer’s instructions (QIAGEN) with the following alterations. For the
immunization of rabbits, cells expressing pMS254 were lysed in lysis buffer (6
M guanidine-HCl, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0, and 5 mM imida-
zole), and lysates were applied to a column containing 2 ml of Ni-NTA
agarose. The column was washed with lysis buffer containing 6 M urea
instead of guanidine-HCl and with urea buffer containing 30 mM imidazole
instead of 5 mM. Proteins were eluted with 200 mM imidazole in urea buffer.
For native proteins used for cross-linking studies, lysis, washing, and elution
was done with imidazole concentrations as described above, but in 50 mM
sodium-phosphate, 300 mM NaCl, pH 8.0.
The coding region of VIPP1 was amplified by PCR from cDNA clone
GTTCTCTCGC-3? and T7. The ?1.35-kb PCR product was digested with SapI
and XhoI and ligated into SapI-XhoI–digested pTYB11 (NEB, Frankfurt, Ger-
many), giving pMS319. pMS319 was expressed in E. coli ER2566 and purified
by chitin affinity chromatography according to the manufacturer’s instruc-
tions (NEB). Pure VIPP1 was dialyzed extensively at 4°C against KMH buffer
(20 mM HEPES-KOH, pH 7.2, 80 mM KCl, and 2.5 mM MgCl2). The calculated
masses matched the masses determined for the purified proteins by mass
spectrometry with a deviation of ? 0.01%.
Purification of HSP70B from Chlamydomonas
One liter of HSP70B-overexpressing strain CF184 (Schroda et al., 1999) was
grown to a density of ?8 ? 106cells/ml, harvested by centrifugation, and
resuspended in a final volume of 10 ml of KMH buffer. Cells were supple-
mented with FCCP to a final concentration of 10 ?M and incubated on a
shaker for 15 min at 25°C. After addition of 0.25? protease inhibitor cocktail,
cells were sonicated on ice for 90 s. The lysate was precleared by a 20-min
centrifugation at 38,000 ? g and 4°C, and the supernatant was loaded onto a
sucrose cushion (20 mM HEPES-KOH, pH 7.2, 0.4 M sucrose) and centrifuged
in a TI50 rotor for 30 min at 152,000 ? g and 4°C. The supernatant was
supplied with Triton-X 100 to a final concentration of 0.1% and stored at
About 1 mg of CGE1 containing N- and C-terminal hexahistidine tags
(Schroda et al., 2001b) was purified from an overexpressing E. coli strain by
nickel-NTA chromatography. Purified CGE1 was incubated in KMH with a
10- ? 5-cm nitrocellulose membrane (Amersham Biosciences) overnight at
4°C, washed once with KMHT (KMH, 0.1% Triton X-100), blocked for 2 h with
5% nonfat dry milk in KMHT at 25°C, and washed 3 ? 10 min with KMHT
at 25°C. The membrane was incubated with the CF184 lysate for 1 h at 25°C
and washed 3 ? 10 min with KMHT and 2 ? with KMH. HSP70B was eluted
by incubating the membrane with 10 ml of KMH containing 5 mM ATP for 20
min, concentrated by centrifugation in Amicon Ultra-4 tubes (Millipore, Mol-
sheim, France), and dialyzed against 2 liters of KMH at 4°C overnight. The
yield was ?20 ?g of HSP70B.
CDJ2, CD?2, and HSP70B (2.6 ?M each) were mixed in a buffer containing 14
mM sodium phosphate, pH 8.0, 85 mM NaCl, 28 mM imidazole, 65 mM KCl,
16 mM HEPES, pH 7.2, and 2.2 mM MgCl2and were incubated at 30°C for 10
min. After addition of glutaraldehyde to a final concentration of 0.1%, incu-
bation at 30°C was continued for another 20 min. After that, 1 volume of 2?
Laemmli sample buffer containing 400 mM glycine was added. The mixtures
were incubated at room temperature for 40 min and separated on 4–18%
To obtain cDNAs that encode chloroplast-targeted J-domain
proteins, we searched the Chlamydomonas EST libraries gen-
erated recently (Asamizu et al., 1999, 2000; Shrager et al.,
2003) with the amino acid sequence of the J-domain of E. coli
DnaJ. Two partial cDNA contigs were assembled that po-
tentially encoded J-domain proteins with N-terminal exten-
sions which by the ChloroP program (Emanuelsson et al.,
1999) were predicted to be chloroplast transit peptides. The
corresponding cDNAs AV626034 and AV387908 were se-
quenced to completion (GenBank accession nos. AY387908
and AY696657, respectively). The mature protein potentially
encoded by cDNA AY696656 has a calculated molecular
mass of 40.26 kDa and exhibits 52% identity and 68% simi-
larity to the chloroplast DnaJ homolog PCJ1 identified in pea
(Schlicher and Soll, 1997). In addition to the J-domain, it
contains a glycin/phenylalanin-rich region and a conserved
cysteine cluster predicted to form a zinc finger-like motif
involved in the binding of denatured polypeptides (Szabo et
al., 1996). The gene product was named chloroplast DnaJ
protein 1 (CDJ1), and it is encoded by a gene located on
scaffold 49 (nt 2174–6823) of the 2.0-version of the Chlamy-
chlre2/chlre2.home.html). As judged by its similarity to
DnaJ, CDJ1 is likely to deliver denatured polypeptides to
chloroplast Hsp70(s) for their refolding to the native state
(Szabo et al., 1996). In support for this view, we found CDJ1
mRNA strongly induced by heat shock (Figure 1A). CDJ1
mRNA accumulation peaked in cells exposed to 40°C be-
tween 15 and 30 min; thereafter, CDJ1 mRNA levels de-
creased again. CDJ1 also was induced by a shift of dark-
adapted cells to dim light; here, mRNA levels peaked 45 min
after transfer to light and then decreased again (Figure 1C).
Both heat shock and light induction of CDJ1 closely resem-
bled that of the Chlamydomonas HSP70A-C genes (von Gro-
moff et al., 1989).
cDNA AY696657 encodes a protein of 38.4 kDa, which
according to the TargetP program (Emanuelsson et al., 2000),
is targeted to the chloroplast and processed to a 31.8-kDa
mature form (Figure 2). The J-domain protein encoded by
this cDNA contains neither a glycine/phenylalanine-rich
region nor a zinc finger domain. The gene encoding this
protein is located on scaffold 44 (nt 188990–198382) of the
2.0-version of the Chlamydomonas genome sequence and was
named CDJ2. The 3? untranslated region of CDJ2 transcript
is remarkably short (87 nucleotides) compared with the
average in Chlamydomonas (several hundred nucleotides; Sil-
flow, 1998). CDJ2 mRNA was barely detectable by RNA gel
blot analyses but seemed not to be induced by heat shock
Database searches using the amino acid sequence located
C-terminally to the CDJ2 J-domain revealed potentially chlo-
roplast-targeted CDJ2 homologues in Arabidopsis and rice
(Figure 2) and identified several higher plant ESTs that
potentially encode CDJ2 homologues. In contrast, no CDJ2
homologues were identified in Cyanobacteria or other bac-
teria nor in nonphotosynthetic eukaryotes. Except for the
Vol. 16, March 20051167
J-domain, no conserved motifs were located within the CDJ2
proteins, and no function was yet attributed to them. Within
the C termini of the CDJ2 proteins the COILS program
(Lupas et al., 1991) located two regions that may form coiled-
coil structures known to mediate protein–protein interac-
tions (Figure 2). One of the predicted coiled-coil regions in
Chlamydomonas CDJ2 is interrupted by a 74-amino acid se-
quence stretch rich in serine (27%), proline (20%), and gly-
cine (13.5%). Adjacent to its J-domain, Chlamydomonas CDJ2
contains another 27-amino acid glycine-rich sequence (44%
glycine) that is absent in the Arabidopsis and rice CDJ2 ho-
mologues (Figure 2). Thus, the predicted mature Arabidopsis
and rice proteins are significantly smaller than those of
Chlamydomonas CDJ2 (22–23 vs. 31.8 kDa). The high content
of charged residues and the lack of putative transmembrane
regions suggest that all three CDJ2 homologues are soluble
We conclude that CDJ2 is conserved from algae to higher
plants. It lacks the glycine/phenylalanine-rich region and
the zinc finger domain typical for DnaJ homologues in-
volved in protein folding, but it contains domains able to
mediate protein–protein interactions.
Coimmunoprecipitation of Chlamydomonas VIPP1 with
To gain insight into the biological function of CDJ2, we used
coimmunoprecipitation to identify proteins that interact
with CDJ2. For this, a polyclonal antibody was raised
against mature CDJ2 expressed in E. coli. In protein gel blot
analyses of whole cell and soluble Chlamydomonas proteins,
the CDJ2 antibody recognized a single band at ?32 kDa
(Figure 1B), which correlated well with the 31.8 kDa calcu-
lated for the mature protein. With the CDJ2 antibody, we
could verify our notion from RNA gel blots that CDJ2 is not
a heat shock-induced protein (Figure 1B).
One hundred microliters of CDJ2 antiserum was sufficient
to precipitate quantitatively all CDJ2 protein from soluble
extracts from ?1010Chlamydomonas cells (Figure 3). Silver
staining of the proteins immunoprecipitated with the CDJ2
antibody revealed a protein of ?28 kDa that coprecipitated
with CDJ2 at about equal quantities. Subsequent immuno-
detection revealed that this ?28-kDa protein was not recog-
nized by the CDJ2 antibody (Figure 3). The CDJ2 preim-
mune serum precipitated an unknown ?26-kDa protein, but
no CDJ2. Bands corresponding to CDJ2 and the ?28-kDa
CDJ2 coprecipitate were excised, digested with trypsin, and
analyzed by mass spectrometry. Three peptides were iden-
tified for CDJ2 (Figure 2). For the ?28-kDa coprecipitate, we
found one peptide encoded by EST AV632440, which codes
for a Chlamydomonas homolog of the VIPP1.
The Chlamydomonas VIPP1 Gene and Gene Product
We sequenced the VIPP1 cDNA corresponding to EST
AV632440 (GenBank accession no. AY696658) and found
that the VIPP1 gene is located on scaffold 23 (nt 582575–
587082) of the 2.0-version of the Chlamydomonas genome
sequence. In RNA gel blot analyses, we observed an approx-
imately threefold induction of VIPP1 transcript after heat
shock (Figure 1A) and an approximately fivefold induction
after dark-to-light shift (Figure 1C). Chlamydomonas VIPP1
shares ?50% identical and ?73% similar residues with
VIPP1 homologues from pea, Arabidopsis, and rice, which in
turn share ?78% identical and ?90% similar residues with
each other. Chlamydomonas and higher plant VIPP1 proteins
all contain N-terminal extensions that according to ChloroP
represent chloroplast transit peptides. From the cleavage site
predicted by TargetP, mature Chlamydomonas VIPP1 has a
size of 28.4 kDa. Accordingly, a polyclonal antibody raised
against mature Chlamydomonas VIPP1 expressed in E. coli
detected a protein of ?28 kDa (Figure 1B). Despite the
increase of VIPP1 mRNA after heat shock and dark-to-light
dark-to-light shift. (A) CDJ1 and VIPP1 mRNA accumulation after
shift of cells from 25 to 40°C. RNA gel blots with 10 ?g of total RNA
per lane were hybridized with probes for CDJ1, VIPP1, and the
Chlamydomonas ?-like protein 2 (c?lp2). The constitutively expressed
c?lp2 served as loading control. (B) CDJ2 and VIPP1 protein accu-
mulation after shift of cells from 25 to 40°C. Protein samples were
from the same experiment depicted in A. Gels were loaded on the
basis of equal chlorophyll concentrations (2 ?g/lane). Constitu-
tively expressed Cytf served as loading control. (C) CDJ1 and VIPP1
mRNA accumulation after shift of cells from 16-h dark to dim light.
RNA gel blots with 10 ?g of total RNA per lane were hybridized
with the same probes used in A.
Induction of CDJ1, CDJ2, and VIPP1 after heat shock and
C. Liu et al.
Molecular Biology of the Cell 1168
shift, we observed no significant changes on VIPP1 protein
level after these treatments (Figure 1B; unpublished data).
Intraplastidal Localization of Chlamydomonas VIPP1 in
Stroma, Thylakoids, and Low-density Membranes
In previous studies, VIPP1 in Cyanobacteria (Westphal et al.,
2001), pea (Li et al., 1994), and Arabidopsis (Kroll et al., 2001)
was found exclusively in membrane fractions. Because we
used soluble extracts for our immunoprecipitations, the
identification of VIPP1 from these was unexpected. To ad-
dress this discrepancy, we performed a crude fractionation
of Chlamydomonas cells into membranes and soluble pro-
teins. For this, a cell wall-deficient strain was ruptured by
freeze-thawing or sonication, and soluble proteins were sep-
arated from membranes by a 355,000 ? g centrifugation. The
VIPP1 antibody detected a ?28-kDa protein present in ap-
proximately equal amounts in soluble and membrane frac-
tions of freeze-thawed and sonicated cells (Figure 4A). Be-
cause fractions were loaded on a volume basis, the data
suggest that about one-half of the Chlamydomonas VIPP1
protein is soluble and the other half is membrane-associated.
In contrast, only little HSP70B and CDJ2 were detected in the
membrane fractions (Figure 4A).
Next, we set out to verify the chloroplast localization of
CDJ2 and VIPP1 and to determine which membranes
Chlamydomonas VIPP1 is associated with. Chlamydomonas
chloroplasts were isolated, lysed by hypoosmotic shock, and
separated into stroma, thylakoid, and low-density mem-
brane fractions. Low-density membranes are considered to
consist of inner envelopes and of transitory membranes
between inner envelope and thylakoids (Zerges and Roch-
aix, 1998). In addition, mitochondria were isolated. The pu-
rity of the fractions was tested with antibodies against mi-
tochondrial carboanhydrase, stromal HSP70B, and CGE1
(the nucleotide exchange factor of HSP70B; Schroda et al.,
2001b), and the integral thylakoid membrane protein Cytf.
Chloroplasts were significantly contaminated by mitochon-
dria and mitochondria slightly by thylakoids, as judged by
the detection of carbonic anhydrase in the chloroplast frac-
tion, and Cytf in the mitochondrial fraction, respectively
(Figure 4B). The low-density membranes contained Cytf, but
no mitochondrial carboanhydrase, whereas the thylakoids
were heavily contaminated with carboanhydrase. The
stroma fraction contained no Cytf and therefore was free of
thylakoidal contaminations but contained some mitochon-
drial carboanhydrase (Figure 4B).
CDJ2 was detected in chloroplasts, stroma, and very
weakly in low-density membranes, but it was absent in
thylakoids and mitochondria (Figure 4B). Thus, CDJ2 exhib-
and Chlamydomonas (C.r). Residues highlighted in black are conserved in all three CDJ2 homologues, those highlighted in gray are conserved
only in two of the three. Conserved amino acids are N/Q, D/E, R/K, S/T, F/Y, A/G, and V/I/L/M. Chloroplast transit peptides are
indicated by an interrupted line, and cleavage sites as predicted by the TargetP program are boxed. The conserved J-domains are underlined
with a dotted line, and putative coiled-coil regions as predicted by the COILS program are underlined with solid lines. Sequences of peptides
identified by mass spectrometry from immunoprecipitated Chlamydomonas CDJ2 are given below the alignment. Alignments were made with
the ClustalW program, refined manually and piled up with the GeneDoc program. Accessions for sequences used for the alignment of CDJ2
homologues are NP_200769 (Arabidopsis; atDjB42; Miernyk, 2001), AAO18454 (rice), and AY696657 (Chlamydomonas).
Alignment of CDJ2 homologues. Aligned are amino acid sequences deduced from CDJ2 genes from Arabidopsis (A.t), rice (O.s),
extract from ?1010Chlamydomonas cells were incubated with protein
A-Sepharose coupled to antibodies of either preimmune serum (Pre)
or anti-CDJ2 serum. Precipitated proteins were eluted under reduc-
ing conditions. Proteins were separated on a 7.5–15% SDS-poly-
acrylamide gel and visualized by silver staining (left gel). Ten
microliters of each of the soluble proteins before (Soluble) and after
incubation with preimmune or anti-CDJ2 sera (Soluble after IP) and
the proteins eluted from the immunoprecipitations (IP eluate) was
separated on a 7.5–15% SDS-polyacrylamide gel, transferred to ni-
trocellulose, and immunodecorated with anti-CDJ2 antibodies
(right gel). The positions of IgG heavy chains (HC) and light chains
(LC) are indicated.
Immunoprecipitation of CDJ2. Six milliliters of a soluble
Vol. 16, March 2005 1169
ited exactly the same fractionation pattern as stromal CGE1.
HSP70B also showed the same fractionation pattern as CGE1
but also was detected in low-density membranes and thyla-
koids, corroborating previous reports (Schroda et al., 2001b;
Friso et al., 2004). VIPP1 was detected in chloroplasts, low-
density membranes, stroma, thylakoids, and weakly in mi-
tochondria (Figure 4B). The presence of some VIPP1 in mi-
tochondria most likely is due to their slight contamination
by thylakoids. Therefore, VIPP1 exhibited a combination of
the fractionation patterns of Cytf and CGE1. Note that in
independent fractionation experiments, the low-density
membrane preparation was devoid of Cytf, but it still con-
tained VIPP1 and some HSP70B. In addition to the major
?28-kDa protein, the VIPP1 antibody also detected a minor
protein at ?26 kDa in whole cells, chloroplasts, and low-
density membranes, which by mass spectrometry was re-
vealed to be a truncated form of VIPP1 (Figure 4B, asterisk).
Also in pea and Arabidopsis, two VIPP1 forms with identical
N termini, but a size difference of ?2 kDa, were detected
(Kroll et al., 2001). The functional significance of these two
VIPP1 forms is unclear.
In summary, our fractionation experiments indicate that
CDJ2 and VIPP1 indeed are chloroplast proteins. Both seem
to be localized in the stroma. VIPP1 also was located to
low-density membranes and thylakoids.
Coimmunoprecipitation of CDJ2 with Anti-VIPP1
The size of the ?28-kDa protein that coprecipitated with
CDJ2 matched well with that predicted for VIPP1. However,
the identification of only a single VIPP1 peptide is not suf-
ficient to conclude that the ?28-kDa CDJ2 coprecipitate is
indeed VIPP1. To verify VIPP1 as a CDJ2 interaction partner,
we used the VIPP1 antibody for the immunodetection of
CDJ2 immunoprecipitations. As shown in Figure 5B, the
VIPP1 antibody clearly recognized the major ?28-kDa pro-
tein that coprecipitated with CDJ2. Moreover, when the
VIPP1 antibody was used for immunoprecipitation of VIPP1
from soluble extracts, among others a minor coprecipitating
protein of ?32 kDa was detected by silver staining (Figure
5A). Immunodetection with CDJ2 antibodies identified this
?32-kDa protein as CDJ2 (Figure 5B). Several proteins in the
?40- to 60-kDa range, which were precipitated by the VIPP1
preimmune serum were not detected by either CDJ2 or
VIPP1 antibodies. Interestingly, whereas anti-CDJ2 antibod-
ies precipitated CDJ2 and VIPP1 in about equal amounts
(Figures 3 and 5A), the anti-VIPP1 antibody coprecipitated
only little CDJ2 (Figure 5A). Immunoprecipitation of VIPP1
from membranes solubilized with 2% Triton X-100 copre-
cipitated hardly any CDJ2 (Figure 5, A and B).
VIPP1 protein that was precipitated with anti-VIPP1 an-
tibodies migrated as a double band at ?28 kDa in SDS-gels
(Figure 5A, asterisk). The double band consisted of a major
and a minor band, the minor band migrating slightly faster
than the major one. In contrast, VIPP1 that was coprecipi-
tated with anti-CDJ2 antibodies seemed to consist only of
the major, slow-migrating VIPP1 (Figure 5, A and B). Ap-
parently, part of the VIPP1 pool is modified such that its
migration is altered. To test, whether modified VIPP1 corre-
sponds to the upper or to the lower band, we separated the
VIPP1 protein that we had overexpressed and purified from
E. coli for the generation of antibodies on the same gel next
to the VIPP1 immunoprecipitations (Figure 5B). VIPP1 was
expressed as a fusion protein that after cleavage had alanine
38 as N-terminal amino acid. Alanine 38 aligns with methi-
onine 61, which has been identified as the N-terminal amino
acid of mature pea VIPP1 (Westphal et al., 2001). VIPP1 that
was purified from E. coli and verified by mass spectrometry
to be nonmodified comigrated with the major ?28-kDa
VIPP1 band (Figure 5B). Therefore, the major ?28-kDa
VIPP1 band is likely to correspond to the unmodified pro-
tein and the faster migrating form seems to contain a mod-
ification that increases its migration properties in SDS-poly-
acrylamide gels, or simply is a VIPP1 degradation product.
domonas cell wall-deficient cells were sonicated (Son) or ruptured by
freeze-thawing (F/T) and separated into soluble (S) or membrane-
enriched (M) fractions on a volume basis. Proteins from whole cells
(WC, 14 ?g) and fractions (S, 4 ?g; M, 10 ?g) were separated on a
7.5–15% SDS-polyacrylamide gel, transferred to nitrocellulose, and
immunodecorated with antibodies against HSP70B, CDJ2, and
VIPP1. (B) Chlamydomonas chloroplasts (Cp) were isolated, lysed by
hypoosmotic shock and separated into stroma (St), low-density
membranes (LM) and thylakoid membranes (Th). Mitochondria
(Mt) were isolated from the same strain. Proteins from whole cells
(WC) and fractions (7 ?g each) were separated on a 7.5–15% SDS-
polyacrylamide gel, transferred to nitrocellulose, and immunodeco-
rated with antibodies against HSP70B, Cytf, CDJ2, VIPP1, CGE1,
and mitochondrial carbonic anhydrase (CA).
Intracellular localization of CDJ2 and VIPP1. (A) Chlamy-
C. Liu et al.
Molecular Biology of the Cell 1170
In summary we show that the ?28-kDa protein coprecipi-
tating with CDJ2 is indeed VIPP1 and vice versa that CDJ2
coprecipitated with VIPP1.
Coimmunoprecipitation of HSP70B with CDJ2 and VIPP1
Because J-domain proteins are known to present bound
proteins to a specific Hsp70 chaperone (Cyr et al., 1994), we
wondered which Hsp70 may take over VIPP1 apparently
presented by CDJ2. In the silver-stained gel shown in Figure
5A, one protein in the 70-kDa range was observed that
specifically coprecipitated with CDJ2. A protein of the same
size coprecipitated with VIPP1 but in much larger amounts.
With VIPP1 a second ?66-kDa protein of more diffuse mi-
gration pattern also was coprecipitated, which seemed to be
absent in the CDJ2 precipitate. The two ?66- and ?70-kDa
bands were excised, digested with trypsin, and analyzed by
mass spectrometry. The protein in the ?70-kDa band was
clearly identified as HSP70B: four HSP70B peptides were
detected from the ?70-kDa protein that coprecipitated with
CDJ2, and 18 HSP70B peptides were identified from the
?70-kDa protein that coprecipitated with VIPP1. In addi-
tion, a specific HSP70B antibody detected this band in both
precipitates (Figure 5B). Surprisingly, eight HSP70C pep-
tides (the mitochondrial Hsp70 homolog; Schroda, 2004)
were detected from the ?66-kDa protein that coprecipitated
with VIPP1. Because the immunoprecipitations were per-
formed from whole cell extracts, we believe that native
VIPP1 may expose Hsp70-binding motifs that also are rec-
ognized by other DnaK-type chaperones, like mitochondrial
When the HSP70B antibody was used in a reciprocal
experiment to immunoprecipitate HSP70B from soluble pro-
teins, VIPP1, and at low concentrations also CDJ2 coprecipi-
tated with HSP70B (Figure 5C). Whereas the major stable
interaction partner of CDJ2 seems to be VIPP1, VIPP1 seems
to be mostly interacting with HSP70B. Note that membrane-
associated VIPP1 only interacted with negligible amounts of
both, CDJ2 and HSP70B (Figure 5, A and B).
HSP70s are known to bind substrate proteins with high
affinity in the ADP state and with low affinity in the ATP
state (Bukau and Horwich, 1998). To test whether the
HSP70B–VIPP1 interaction is influenced by the ATP concen-
tration, we immunoprecipitated VIPP1 from ATP-supple-
mented extracts or from extracts prepared from ATP-de-
pleted cells. As shown in Figure 6, in cell extracts depleted
from ATP, the interaction of VIPP1 with HSP70C and
HSP70B was comparable with that observed in extracts from
nontreated cells (Figure 5), indicating that the ATP concen-
trations in our cell extracts were low. In contrast, in ATP-
supplemented extracts, the VIPP1 interaction with HSP70C
was abolished and that with HSP70B was significantly re-
duced (Figure 6). This suggests that VIPP1 has a higher
affinity for HSP70B than for HSP70C. Interestingly, CDJ2
that was immunoprecipitated from ATP-supplemented cell
extracts coprecipitated significantly less VIPP1 and HSP70B.
The identity of the ?45-kDa protein that also coprecipitated
with CDJ2 in an ATP-dependent manner (Figure 6) could
not yet be revealed.
Together, the data suggest that the HSP70 partner of CDJ2
is stromal HSP70B and that native VIPP1 behaves like a
substrate for HSP70B. The interaction of VIPP1 with CDJ2
and HSP70B is significantly weaker in the presence of ATP
but apparently not abolished.
Chlamydomonas soluble extract from ?1.5 ? 1010cells was incubated
with protein A-Sepharose coupled to antibodies of either preim-
mune (Pre), anti-CDJ2, or anti-VIPP1 serum, and membranes solu-
bilized with Triton X-100 (Mem) were incubated with anti-Vipp1
antibodies coupled to protein A-Sepharose. Precipitated proteins
were separated on a 7.5–15% SDS-polyacrylamide gel under nonre-
ducing conditions and visualized by silver staining. The asterisk
indicates a slightly faster migrating VIPP1 form. (B) Proteins from
the immunoprecipitation shown in A in addition to heterologously
expressed CDJ2 (containing N- and C-terminal hexahistidine tags)
and VIPP1 were separated on a 7.5–15% SDS-polyacrylamide gel,
transferred to nitrocellulose, and immunodecorated with antibodies
against HSP70B, CDJ2, and VIPP1. Note that the CDJ2 detection was
exposed much longer than the VIPP1 detection. (C) Chlamydomonas
total soluble protein (Sol) was incubated with protein A-Sepharose
coupled to antibodies of Pre or anti-HSP70B serum. Total soluble
and precipitated proteins were separated on a 7.5–15% SDS-poly-
acrylamide gel, transferred to nitrocellulose, and immunodecorated
with antibodies against HSP70B, CDJ2, and VIPP1.
Immunoprecipitation of CDJ2, VIPP1, and HSP70B. (A)
Vol. 16, March 20051171
Verification of the Interaction between CDJ2 and HSP70B
by Glutaraldehyde Cross-linking
Han and Christen (2003) provided evidence that DnaJ and
DnaK may bind to different sites of the same substrate
molecule. Thus, if this was the case also for CDJ2 and
HSP70B binding to VIPP1, HSP70B may have coprecipitated
with CDJ2 via VIPP1 without necessarily being involved in
a common chaperoning process. To rule out this possibility,
we had to confirm that CDJ2 and HSP70B also interacted
physically in the absence of VIPP1. To test this, we per-
formed in vitro glutaraldehyde cross-linking studies with
purified HSP70B and CDJ2. To assay the role of the J-domain
in this interaction, we also included a CDJ2 derivative lack-
ing the N-terminal J-domain (CD?2). Glutaraldehyde cross-
linking has been used successfully to monitor complex for-
mation of Hsp70 and its cochaperones (Wu et al., 1996; Azem
et al., 1997).
As judged by its inability to interact with the CGE1 co-
chaperone in glutaraldehyde cross-linking experiments,
HSP70B heterologously expressed in E. coli seemed to be
nonfunctional (Willmund and Schroda, unpublished re-
sults). Thus, we purified the HSP70B protein from Chlamy-
domonas-soluble extracts by using the CGE1 cochaperone as
an affinity matrix. Silver staining of the purified proteins
after separation on an SDS-polyacrylamide gel revealed that
HSP70B and CDJ2 contained few impurities, whereas the
preparation of CD?2, for which the expression level in E. coli
was very low, contained several impurities (Figure 7A).
The purified proteins were incubated alone or in equimo-
lar amounts with their partner, cross-linked with glutaral-
dehyde, separated on SDS-polyacrylamide gels, transferred
to nitrocellulose, and immunodecorated with antibodies
against CDJ2 or HSP70B. CDJ2, CD?2, and HSP70B alone
were present mainly as monomers (Figure 7B, lanes 1, 2, and
5). When incubated with HSP70B, CDJ2 formed a major
complex of ?95 kDa and CD?2 a minor one of ?85 kDa with
HSP70B (Figure 7B, lanes 3 and 4). HSP70B incubated with
CDJ2 shifted to a complex of ?95 kDa but also formed
high-molecular-weight polymers that hardly entered the gel
(Figure 7B, lane 6). When incubated with CD?2, HSP70B
smeared weakly into a ?85-kDa complex but did not form
polymers (Figure 7B, lane 7).
In summary, our data suggest that CDJ2 and CD?2 both
interact physically as monomers with monomeric HSP70B.
The weak ?85-kDa HSP70B–CD?2 complex may arise from
a specific interaction but also from the recognition of CD?2
by HSP70B as a substrate due to misfolding of CD?2 in-
duced by the absence of its J-domain. Compared with the
weak HSP70B–CD?2 complex,
HSP70B–CDJ2 complex points to an important role of the
CDJ2 J-domain for mediating the interaction of the two
proteins. CDJ2’s J-domain also is required to induce poly-
merization of HSP70B.
Analysis of Soluble CDJ2- and VIPP1-containing
Complexes by BN-PAGE
We intended to analyze the complexes formed by soluble
VIPP1 and CDJ2. For this, we separated native protein com-
plexes from soluble Chlamydomonas extracts by size by using
BN-PAGE and transferred the complexes directly to nitro-
cellulose membranes. Immunodetection with the CDJ2 anti-
body revealed a strong signal ?66 kDa from monomeric or
dimeric CDJ2 and weaker signals from complexes of ?150
and ?300 kDa (Figure 8). The VIPP1 antibody gave a strong
signal at ?66 kDa from monomeric or dimeric VIPP1 and
weak ones from complexes at ?150, ?180, ?300, and ? ?669
kDa. A signal in the 550-kDa region seems to be a cross-
reaction of the VIPP1 antibody with native Rubisco, which
migrates in large quantities at this position.
tion. Soluble extract from Chlamydomonas cells that were depleted
from ATP or repleted with 2.5 mM Mg-ATP was incubated with
protein A-Sepharose coupled to antibodies of either preimmune
(Pre), anti-CDJ2, or anti-VIPP1 serum. Precipitated proteins were
separated on a 7.5–15% SDS-polyacrylamide gel under nonreducing
conditions and visualized by silver staining.
ATP dependence of the VIPP1–CDJ2–HSP70B interac-
CDJ2 by glutaraldehyde cross-linking. (A) HSP70B purified from
Chlamydomonas and heterologously expressed CDJ2 and CDJ2 lack-
ing its J-domain (CD?2) (both containing N- and C-terminal hexa-
histidine tags) were separated on a 7.5–15% SDS-polyacrylamide gel
and visualized by silver staining. (B) CDJ2, CD?2, and HSP70B (2.6
?M each) were cross-linked, separated on 4–18% SDS-polyacryl-
amide gels, transferred to nitrocellulose, and immunodecorated
with antibodies against CDJ2 or HSP70B.
In vitro analysis of the interaction between HSP70B and
C. Liu et al.
Molecular Biology of the Cell1172
CDJ2 in the second dimension was found mostly below 66
kDa and in a complex of ?150 kDa (Figure 8). A minor
complex was also observed in a region ? ?669 kDa, which
was not detected in the first dimension. In contrast, the
?300-kDa complex detected in the first dimension was ab-
sent in the second dimension, a finding we have reproduced
many times. Either the CDJ2 antibody cross-reacts only with
the native form of a protein in this ?300-kDa complex or
CDJ2 in this complex resists SDS-denaturation and does not
enter the second dimension gel. In SDS-gels, some CDJ2
protein reproducibly migrated slightly slower than bulk
CDJ2 (Figures 3, 5B, and 8). It is not clear whether this is due
to a modification of CDJ2 or due to incomplete denaturation.
All VIPP1 complexes detected in the first dimension also
were detected at similar intensities in the second dimension
(Figure 8). With the HSP70B antibody, no distinct complexes
but a smear into the higher molecular weight region was
detected (our unpublished data; Schroda et al., 2001b). Ex-
actly the same pattern of CDJ2- and VIPP1-containing com-
plexes was detected when cells were lysed more gently by
vortexing with glass beads instead of sonication (our unpub-
lished data). This argues against the possibility of a nonspe-
cific disassembly of the ? ?669-kDa VIPP1/CDJ2 complex by
the harsh sonication procedure. When Chlamydomonas VIPP1
purified from E. coli was separated by BN-PAGE, VIPP1 was
detected only at positions ?66 and ? ?669 kDa (our unpub-
lished data). This indicates that VIPP1 alone has the capa-
bility to form large oligomers and thus that the VIPP1 signal
at ? ?669 kDa is not just low-molecular-weight VIPP1 asso-
ciated with membrane vesicles, which may still be present in
the soluble cell extracts and would hardly enter the native
In summary, we can conclude that 1) most of soluble
VIPP1 and CDJ2 exist as monomers or dimers; 2) VIPP1
alone can assemble into oligomers of ? ?669 kDa; and 3)
CDJ2 and VIPP1 may form heterodimers and/or complexes
of ?150, ? ?669, and perhaps ?300 kDa.
We report on the identification of two cDNAs that encode
the CDJ1 and CDJ2 proteins. We show that the CDJ1 gene is
strongly induced by heat shock and slightly by light (Figure
1) and potentially encodes a plastidic member of the DnaJ
family involved in protein folding (Szabo et al., 1996). A
plastidic DnaJ homologue, named PCJ1, has previously been
reported in pea chloroplasts (Schlicher and Soll, 1997).
CDJ2 and HSP70B Form a Plastidic Chaperone Pair
For CDJ2, the following findings suggest that it forms a
plastidic chaperone pair with HSP70B. 1) From soluble cell
extracts, HSP70B coimmunoprecipitated with CDJ2, and
vice versa; CDJ2 coprecipitated with HSP70B (Figure 5). 2)
Both proteins colocalize to the chloroplast stroma (Figure
4B), to which at most two HSP70 chaperones are located,
with HSP70B being the most prominent (Schroda, 2004). 3)
In vitro cross-linking studies revealed that CDJ2 forms a
complex with HSP70B in a 1:1 stoichiometry and that CDJ2
can catalytically induce polymerization of HSP70B into
high-molecular-weight complexes without being part of
these (Figure 7B). Stable interactions between J-domain pro-
teins and Hsp70s already have been reported for some chap-
erone pairs, e.g., mammalian hsp40/Hsp70 (Sugito et al.,
1995), ER Sec63p/BiP (Brodsky and Schekman, 1993), and
bovine brain auxilin/Hsc70 (Jiang et al., 1997), but not for the
E. coli DnaJ/DnaK couple (King et al., 1995). Even catalytic
polymerization by J-domain proteins has been demon-
strated for HSP70s of the eukaryotic cytosol (King et al.,
1995; Jiang et al., 1997), but again not for bacterial DnaK
(King et al., 1999). Therefore, our results show that polymer-
ization also of a prokaryotic-type Hsp70 (stromal HSP70B)
may be induced by the J-domain protein CDJ2.
According to a model developed previously to explain
Hsp70 polymerization by J-domain proteins (King et al.,
1999), the J-domain protein in the absence of its natural
substrate instead binds an Hsp70 and presents it to the
substrate-binding domain of another Hsp70. The latter un-
dergoes ATP hydrolysis and binds the presented Hsp70 like
a substrate. Bound Hsp70 may in turn bind another Hsp70
newly presented by a J-domain protein, eventually produc-
ing a chain of polymerized Hsp70s. This model implies that
the respective J-domain protein is capable of binding Hsp70
in a region distinct from the J-domain. In support of this
model, in our cross-linking studies we have indeed observed
an interaction of CD?2 (CDJ2 with deleted J-domain) with
HSP70B (Figure 7B). Whether Hsp70 polymerization occurs
in vivo and what its role may be are unclear (King et al.,
VIPP1 Is a Major Target Protein of the HSP70B/CDJ2
We found that soluble VIPP1 is a major target protein of the
HSP70B/CDJ2 chaperone pair. Antibodies against both
CDJ2 and HSP70B coimmunoprecipitated VIPP1, and vice
versa; antibodies against VIPP1 coprecipitated CDJ2 and
HSP70B (Figure 5). Several lines of evidence indicate that the
interactions between these three proteins are specific and
not due to, e.g., a nonspecific recognition of nonnative VIPP1
by the HSP70B/CDJ2 chaperone pair. 1) In contrast to CDJ1,
CDJ2 is induced neither by heat stress nor does it contain
domains typical for DnaJ proteins involved in protein fold-
ing (a glycine/phenylalanine-rich region or a zinc finger-
forming cysteine cluster). Thus, CDJ2 is more likely a J-
domain protein specialized for a specific cellular function
other than protein folding. 2) VIPP1, an unknown ?45-kDa
protein, and HSP70B in this order are the most prominent
interaction partners of CDJ2 (Figure 6). If CDJ2 had a ten-
dency to nonspecifically interact with other proteins, more
of them would have been expected. 3) Recent studies have
provided evidence that E. coli DnaK and DnaJ bind to dif-
Chlamydomonas total soluble proteins (Sol) were separated on a
6–15% native gel (BN-PAGE) and transferred directly to nitrocellu-
lose (top two gels) or separated in the second dimension on a 10%
SDS-PAG and then transferred to nitrocellulose (bottom two gels).
Membranes were immunodecorated with antibodies against CDJ2
and VIPP1. Arrows indicate the positions of complexes that contain
CDJ2, VIPP1, or both.
Analysis of CDJ2 and VIPP1 complexes by BN-PAGE.
Vol. 16, March 20051173
ferent sites on the same substrate protein (Han and Christen,
2003). If this holds for J-domain protein/HSP70 chaperone
pairs in general, the binding of a specialized J-domain pro-
tein and of its HSP70 partner to the same polypeptide sug-
gests specificity. 4) CDJ2 and VIPP1 both contain regions
predicted to form coiled-coils (Figure 2). 5) CDJ2, VIPP1, and
HSP70B all are localized in the same compartment, i.e., the
chloroplast stroma (Figure 4). 6) The interaction of CDJ2
with VIPP1 and with an ?45-kDa protein is ATP dependent,
i.e., less of them interact with CDJ2 in the presence of Mg-
ATP (Figure 6). This observation is consistent with the
model that J-domain proteins deliver bound substrates to
their HSP70 partner in the ATP state, trigger ATP hydrolysis
and dissociate once the substrate is processed by HSP70
(e.g., folded to the native state or translocated across a
membrane) (Bukau and Horwich, 1998). In the absence of
ATP, the J-domain protein keeps binding its substrate, be-
cause HSP70 cannot process it further. Thus, a protein non-
specifically bound to a J-domain protein should remain
bound also in the ATP-state, because it is unlikely that the
HSP70 partner is competent for further processing. 7) Anti-
bodies against chloroplast-targeted HSP90, bacterial GroEL,
and bacterial ClpB detected proteins of the expected molec-
ular weights in soluble extracts, but neither chaperone was
detected in CDJ2 or VIPP1 immunoprecipitates (our unpub-
lished data), suggesting that CDJ2 and VIPP1 specifically
interact only with HSP70 chaperones. 8) Plastidic HSP70B
has a higher affinity for VIPP1 than obviously nonspecifi-
cally binding mitochondrial HSP70C: even in the presence of
ATP HSP70B exhibited some affinity for VIPP1, whereas
HSP70C released VIPP1 entirely (Figure 6). 9) Immunopre-
cipitates prepared with antibodies against stromal CF1? or
CF1? did not contain HSP70B or CDJ2 (our unpublished
data), suggesting that the chaperone pair does not simply
interact with any stromal protein.
VIPP1 is likely to have evolved from duplication of the
pspA gene in Cyanobacteria (Westphal et al., 2001). Because
we found CDJ2 homologues only in algae and higher plants
but not in Cyanobacteria, CDJ2 as mediator for the VIPP1–
HSP70 interaction may have evolved after the endosymbi-
otic event. Perhaps CDJ2 has evolved to link VIPP1 to
DnaK2, the ancestor of today’s stromal Hsp70s, so that a
specific cyanobacterial DnaK system (DnaK1 or DnaK3) may
have become redundant and for this reason got extinct?
Chlamydomonas VIPP1 Is Located to the Stroma and to
The coprecipitation of VIPP1 from soluble cell extracts was
unexpected, because localization studies in pea (Li et al.,
1994) and Arabidopsis (Kroll et al., 2001) detected VIPP1 in
thylakoids and inner envelopes, and in Synechocystis VIPP1
was found exclusively in plasma membranes (Westphal et
al., 2001). However, some pea VIPP1 that cofractionated
with outer envelopes was interpreted as soluble, particulate
VIPP1 with a density similar to that of outer envelopes (Li et
al., 1994). In addition, the probable VIPP1 ancestor PspA was
detected in the cytosol and the inner membrane of E. coli in
about equal quantities (Brissette et al., 1990; Kleerebezem
and Tommassen, 1993). The same localization pattern as for
PspA also was observed for cyanobacterial VIPP1 expressed
in E. coli (DeLisa et al., 2004). Careful fractionation of Chlamy-
domonas cells verified that about one-half of the VIPP1 pro-
tein is indeed in the soluble stroma fraction, whereas the
other half is located to thylakoids and to low-density mem-
branes, which to a large part consist of inner envelopes
(Zerges and Rochaix, 1998) (Figure 4). It is not clear whether
these rather diverse localization patterns of VIPP1 proteins
in different organisms are due to the preparation techniques,
differences in cell physiology, or rather to organism-specific
differences in the affinity of VIPP1 for thylakoids and inner
VIPP1 and CDJ2 Form Distinct Complexes
PspA in E. coli has been demonstrated to form dimers and
perhaps higher order oligomers (Dworkin et al., 2000) and
recently, PspA in vitro has been shown to form oligomeric
rings of 1023 kDa that probably consist of 36 PspA subunits
organized in ninefold symmetry (Hankamer et al., 2004). By
BN-PAGE, we found VIPP1 in soluble cell extracts as mono-
mers or dimers ?66 kDa and in complexes of ?150, ?180,
?300, and ? ?669 kDa. We hypothesize that the ? ?669-kDa
VIPP1 complex found in soluble cell extracts and in purified
VIPP1 samples also may represent large oligomeric rings.
This view is consistent with immunogold studies with
VIPP1 antibodies, in which gold particles were found in
clusters (Li et al., 1994). CDJ2, too, was found in monomers
or dimers ?66 kDa and in complexes of ?150, ?300, and
? ?669 kDa (Figure 8). Thus, CDJ2 and VIPP1 may interact as
heterodimers and/or in the ?150-, ?300-, and ? ?669-kDa
complexes. Immunoprecipitation of CDJ2 led to the copre-
cipitation of approximately equal amounts of VIPP1 (Fig-
ures 3, 5, and 6), whereas immunoprecipitation of VIPP1
coprecipitated only small amounts of CDJ2 (Figure 5). Thus,
in the stroma, either CDJ2 is much less abundant than VIPP1
and is quantitatively organized into complexes with VIPP1,
or few CDJ2 proteins interact with larger VIPP1 oligomers.
The finding that very little CDJ2 and almost stoichiometric
amounts of HSP70B coprecipitated with soluble VIPP1 (Fig-
ures 5 and 6) suggests that CDJ2 may prime VIPP1 for the
binding of HSP70B and in the presence of ATP seems to
leave the HSP70B–VIPP1 complex once it has been estab-
lished (Figure 6).
VIPP1 May Play Roles in Maintaining Membrane
Integrity and/or in Vesicle Traffic
To discuss which cellular function the interaction of HSP70B
and CDJ2 with VIPP1 may have, we first need to summarize
briefly what is known about VIPP1. VIPP1 underexpression
resulted in distorted thylakoids in Arabidopsis and Synecho-
cystis, and vesicle budding from the inner envelope of Ara-
bidopsis chloroplasts was abolished (Kroll et al., 2001; West-
phal et al., 2001). Unfortunately, nothing is known about the
mechanisms involved. Expression of the likely VIPP1 ances-
tor pspA is induced by stresses that affect membrane integ-
rity, such as heat, ethanol, hyperosmolarity, pore-forming
phage proteins, or the accumulation of aberrant secretion
pathway intermediates within secretion pores (Brissette et
al., 1990; Kleerebezem and Tommassen, 1993; Jones et al.,
2003). All these stresses are believed to eventually result in a
decreased transmembrane proton motif force (pmf), which
is likely to trigger induction of the psp operon (Adams et al.,
2003). PspA by an unknown mechanism seems to sustain
membrane integrity, thereby maintaining the pmf (Kleerebe-
zem et al., 1996) and ensuring a proper performance of the
Sec (Kleerebezem and Tommassen, 1993) and of the Tat
pathways (DeLisa et al., 2004). Importantly, VIPP1 can sub-
stitute PspA to improve the performance of an overloaded
Tat pathway in E. coli, and vice versa; PspA can improve the
performance of the thylakoidal Tat pathway in vitro (DeLisa
et al., 2004). Still, some specificity for PspA and VIPP1 func-
tion must exist, because both are present in Cyanobacteria,
but cyanobacterial PspA cannot substitute for cyanobacterial
VIPP1 function in vivo (Westphal et al., 2001).
C. Liu et al.
Molecular Biology of the Cell1174
Thus, two possibilities may explain the VIPP1 mutant
phenotype. First, VIPP1, like PspA, may function exclusively
in sustaining membrane integrity but became specialized to
fulfill this task on chloroplast membranes, e.g., as essential
auxiliary factor for lumenal import pathways. The distorted
thylakoid phenotype in VIPP1 mutants may be a conse-
quence of the malfunctioning of lumenal import. Note that
mutants defective in components of the Sec or Tat pathways
display phenotypes that resemble that of VIPP1 mutants
(Settles et al., 1997). The absence of vesicles may be due to a
secondary effect caused by a lack of a thylakoid-derived
signal required to trigger vesicle budding.
Alternatively, VIPP1 may have adopted a function in ad-
dition to that required for sustaining membrane integrity,
i.e., a role in vesicle traffic. As suggested previously (West-
phal et al., 2001), this additional function may be related to
the C-terminal extension of ?30 amino acids present in
VIPP1 but absent in PspA. Perhaps the mechanism by which
PspA sustains membrane integrity became essential for ves-
Hypothetical Roles of the HSP70B/CDJ2 Chaperone Pair
in the Functional Cycle of VIPP1
The mechanism underlying Hsp70 function is the ability of
this chaperone to bind hydrophobic motifs exposed by vir-
tually all unfolded and some native proteins and to induce
conformational changes in an ATP-dependent reaction (Ru ¨-
diger et al., 1997; Schiene-Fischer et al., 2002). The latter may
result in the refolding of denatured proteins, and in the case
of native protein complexes, in complex disassembly. In E.
coli, for example, DnaK monomerizes RepA dimers and
dissociates DnaB helicase-Lambda P complexes (Alfano and
McMacken, 1989; Wickner et al., 1991); in the eukaryotic
cytosol Hsc70 disassembles clathrin cages (Ungewickell et
al., 1995) and primes disassembled clathrin for reassembly
(Jiang et al., 2000).
We can envision that both a folding and a disassembling/
assembling activity of HSP70B may support VIPP1 function.
It is likely that in a first step CDJ2 binds to VIPP1 through
coiled-coil interactions to recruit HSP70B via its J-domain
and to lock HSP70B onto VIPP1. HSP70B may then target
VIPP1 to sites where unfolded membrane proteins jeopar-
dize membrane integrity. Such a targeting function may
account for the finding that HSP70B and CDJ2 interact
mainly with soluble VIPP1 and barely with membrane-as-
sociated VIPP1 (Figure 5). For a more specialized function in
thylakoidal import, HSP70B associated with VIPP1 may ver-
ify the correct folding of lumenal precursors (unfolded pro-
teins for the Sec pathway; correctly folded, monomeric and
cofactor-bound proteins for the Tat pathway), thereby pre-
venting proton leakage potentially caused by improperly
translocating precursors. Accordingly, the induction of the
VIPP1 and HSP70B genes by heat shock and light (Figure 1;
von Gromoff et al., 1989) may account for a need in the
chloroplast to cope with the accumulation of heat-denatured
membrane proteins and to improve lumenal import at trans-
location pores that become crowded after the onset of light,
Alternatively, the HSP70B/CDJ2 chaperone pair may
carry out disassembly and/or assembly of VIPP1 oligomers.
Such a function would be reminiscent of the Hsc70–auxilin–
clathrin triad, where the J-domain protein auxilin specifi-
cally interacts with assembled clathrin on clathrin-coated
vesicles and recruits Hsc70. Hsc70 then drives clathrin dis-
assembly, eventually resulting in the uncoating of clathrin-
coated vesicles (Ungewickell et al., 1995). Like CDJ2, auxilin
interacts rather stably with Hsc70 (Jiang et al., 1997). Hsc70
binds stoichiometrically to clathrin (Ungewickell et al., 1995),
which also seems to hold for HSP70B binding VIPP1 (Figure
5A). Finally, after the uncoating reaction, Hsc70 in the ADP
state forms a stable presteady-state complex with adaptor
proteins and clathrin. After replacement of ADP by ATP,
this complex turns into a structurally different steady-state
complex, which is more labile but persists. In this complex,
clathrin may be primed to interact again with vesicle mem-
branes (Jiang et al., 2000). Apparently, HSP70B also interacts
with VIPP1 more strongly in the ADP state, but affinity is not
lost completely in the ATP state (Figure 6). In analogy to the
auxilin/Hsc70 chaperone pair, CDJ2/HSP70B might disas-
semble and/or assemble VIPP1 oligomers to recycle the
system for another turn of vesicle formation/transport. Al-
ternatively, disassembly/assembly of VIPP1 might be re-
quired for its activation/inactivation in the process of sus-
taining membrane integrity.
Although we currently cannot distinguish between these
possibilities, our finding that HSP70B and CDJ2 specifically
interact with VIPP1 will pave the path for further studies to
elucidate the roles that stromal chaperones play in the bio-
genesis/maintenance of chloroplast membranes in algae
and higher plants.
We thank the Kazusa DNA Research Institute for providing cDNA clones
AV626034, AV387908, and AV632440. We also thank Michael Hippler for help
with mass spectrometry measurements; Francis-Andre ´ Wollman for the an-
tibodies against Cytf, CF1?, and CF1?; Bernd Bukau for the antibodies against
GroEL and ClpB; and Mats Eriksson for the antibody against mitochondrial
carbonic anhydrase. We are grateful to Olivier Vallon and Christoph Beck for
critical reading of the manuscript and acknowledge Christoph Beck for the
support with laboratory equipment. This work was supported by the Deut-
sche Forschungsgemeinschaft (Schr 617/2-1; 4-1; Be 903/12-1; 12-2).
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