To obtain cDNAs that encode chloroplast-targeted J-domain proteins, we searched the Chlamydomonas
EST libraries generated recently (Asamizu et al
; Shrager et al., 2003
) with the amino acid sequence of the J-domain of E. coli
DnaJ. Two partial cDNA contigs were assembled that potentially encoded J-domain proteins with N-terminal extensions which by the ChloroP program (Emanuelsson et al., 1999
) were predicted to be chloroplast transit peptides. The corresponding cDNAs AV626034 and AV387908 were sequenced 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% similarity 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 c
(CDJ1), and it is encoded by a gene located on scaffold 49 (nt 2174–6823) of the 2.0-version of the Chlamydomonas
genome sequence (http://genome.jgi-psf.org/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 (). CDJ1
mRNA accumulation peaked in cells exposed to 40°C between 15 and 30 min; thereafter, CDJ1
mRNA levels decreased 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 (). Both heat shock and light induction of CDJ1
closely resembled that of the Chlamydomonas HSP70A-C
genes (von Gromoff et al., 1989
Figure 1. Induction of CDJ1, CDJ2, and VIPP1 after heat shock and dark-to-light shift. (A) CDJ1 and VIPP1 mRNA accumulation after shift of cells from 25 to 40°C. RNA gel blots with 10 μg of total RNA per lane were hybridized with probes for CDJ1, (more ...)
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 (). 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; Silflow, 1998
mRNA was barely detectable by RNA gel blot analyses but seemed not to be induced by heat shock (unpublished data).
Figure 2. Alignment of CDJ2 homologues. Aligned are amino acid sequences deduced from CDJ2 genes from Arabidopsis (A.t), rice (O.s), and Chlamydomonas (C.r). Residues highlighted in black are conserved in all three CDJ2 homologues, those highlighted in gray are (more ...)
Database searches using the amino acid sequence located C-terminally to the CDJ2 J-domain revealed potentially chloroplast-targeted CDJ2 homologues in Arabidopsis
and rice () and identified several higher plant ESTs that potentially encode CDJ2 homologues. In contrast, no CDJ2 homologues were identified in Cyanobacteria or other bacteria nor in nonphotosynthetic eukaryotes. Except for the 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 interactions (). One of the predicted coiled-coil regions in Chlamydomonas
CDJ2 is interrupted by a 74-amino acid sequence stretch rich in serine (27%), proline (20%), and glycine (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 homologues (). 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 proteins.
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 involved in protein folding, but it contains domains able to mediate protein–protein interactions.
Coimmunoprecipitation of Chlamydomonas VIPP1 with CDJ2 Antibodies
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 (), which correlated well with the 31.8 kDa calculated 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 ().
One hundred microliters of CDJ2 antiserum was sufficient to precipitate quantitatively all CDJ2 protein from soluble extracts from ~1010 Chlamydomonas cells (). 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 recognized by the CDJ2 antibody (). The CDJ2 preimmune 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 identified for CDJ2 (). 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 approximately threefold induction of VIPP1 transcript after heat shock () and an approximately fivefold induction after dark-to-light shift (). 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 (). Despite the increase of VIPP1 mRNA after heat shock and dark-to-light shift, we observed no significant changes on VIPP1 protein level after these treatments (; 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 address this discrepancy, we performed a crude fractionation of Chlamydomonas
cells into membranes and soluble proteins. For this, a cell wall-deficient strain was ruptured by freeze-thawing or sonication, and soluble proteins were separated from membranes by a 355,000 × g
centrifugation. The VIPP1 antibody detected a ~28-kDa protein present in approximately equal amounts in soluble and membrane fractions of freeze-thawed and sonicated cells (). Because 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 4. Intracellular localization of CDJ2 and VIPP1. (A) Chlamydomonas 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 (more ...)
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 membrane fractions. Low-density membranes are considered to consist of inner envelopes and of transitory membranes between inner envelope and thylakoids (Zerges and Rochaix, 1998
). In addition, mitochondria were isolated. The purity of the fractions was tested with antibodies against mitochondrial 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 mitochondria and mitochondria slightly by thylakoids, as judged by the detection of carbonic anhydrase in the chloroplast fraction, and Cytf
in the mitochondrial fraction, respectively (). 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 mitochondrial carboanhydrase ().
CDJ2 was detected in chloroplasts, stroma, and very weakly in low-density membranes, but it was absent in thylakoids and mitochondria (). Thus, CDJ2 exhibited 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 thylakoids, corroborating previous reports (Schroda et al., 2001b
; Friso et al., 2004
). VIPP1 was detected in chloroplasts, low-density membranes, stroma, thylakoids, and weakly in mitochondria (). The presence of some VIPP1 in mitochondria 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 contained 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 revealed to be a truncated form of VIPP1 (, 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 Antibodies
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 sufficient 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 , the VIPP1 antibody clearly recognized the major ~28-kDa protein 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 (). Immunodetection with CDJ2 antibodies identified this ~32-kDa protein as CDJ2 (). 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 antibodies precipitated CDJ2 and VIPP1 in about equal amounts (Figures and ), the anti-VIPP1 antibody coprecipitated only little CDJ2 (). Immunoprecipitation of VIPP1 from membranes solubilized with 2% Triton X-100 coprecipitated hardly any CDJ2 ().
VIPP1 protein that was precipitated with anti-VIPP1 antibodies migrated as a double band at ~28 kDa in SDS-gels (, 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 coprecipitated with anti-CDJ2 antibodies seemed to consist only of the major, slow-migrating VIPP1 (). Apparently, part of the VIPP1 pool is modified such that its migration is altered. To test, whether modified VIPP1 corresponds 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 (). VIPP1 was expressed as a fusion protein that after cleavage had alanine 38 as N-terminal amino acid. Alanine 38 aligns with methionine 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 (). Therefore, the major ~28-kDa VIPP1 band is likely to correspond to the unmodified protein and the faster migrating form seems to contain a modification that increases its migration properties in SDS-polyacrylamide gels, or simply is a VIPP1 degradation product.
In summary we show that the ~28-kDa protein coprecipitating with CDJ2 is indeed VIPP1 and vice versa that CDJ2 coprecipitated with VIPP1.
Coimmunoprecipitation of HSP70B with CDJ2 and VIPP1 Antibodies
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 , 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 migration 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 addition, a specific HSP70B antibody detected this band in both precipitates (). Surprisingly, eight HSP70C peptides (the mitochondrial Hsp70 homolog; Schroda, 2004
) were detected from the ~66-kDa protein that coprecipitated with VIPP1. Because the immunoprecipitations were performed from whole cell extracts, we believe that native VIPP1 may expose Hsp70-binding motifs that also are recognized by other DnaK-type chaperones, like mitochondrial HSP70C.
When the HSP70B antibody was used in a reciprocal experiment to immunoprecipitate HSP70B from soluble proteins, VIPP1, and at low concentrations also CDJ2 coprecipitated with HSP70B (). 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 ().
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 concentration, we immunoprecipitated VIPP1 from ATP-supplemented extracts or from extracts prepared from ATP-depleted cells. As shown in , in cell extracts depleted from ATP, the interaction of VIPP1 with HSP70C and HSP70B was comparable with that observed in extracts from nontreated cells (), indicating that the ATP concentrations 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 reduced (). 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 () 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.
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 performed 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 lacking the N-terminal J-domain (CDΔ2). Glutaraldehyde cross-linking has been used successfully to monitor complex formation 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 results). Thus, we purified the HSP70B protein from Chlamydomonas-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 CDΔ2, for which the expression level in E. coli was very low, contained several impurities ().
Figure 7. In vitro analysis of the interaction between HSP70B and CDJ2 by glutaraldehyde cross-linking. (A) HSP70B purified from Chlamydomonas and heterologously expressed CDJ2 and CDJ2 lacking its J-domain (CDΔ2) (both containing N- and C-terminal hexahistidine (more ...)
The purified proteins were incubated alone or in equimolar amounts with their partner, cross-linked with glutaraldehyde, separated on SDS-polyacrylamide gels, transferred to nitrocellulose, and immunodecorated with antibodies against CDJ2 or HSP70B. CDJ2, CDΔ2, and HSP70B alone were present mainly as monomers (, lanes 1, 2, and 5). When incubated with HSP70B, CDJ2 formed a major complex of ~95 kDa and CDΔ2 a minor one of ~85 kDa with HSP70B (, 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 (, lane 6). When incubated with CDΔ2, HSP70B smeared weakly into a ~85-kDa complex but did not form polymers (, lane 7).
In summary, our data suggest that CDJ2 and CDΔ2 both interact physically as monomers with monomeric HSP70B. The weak ~85-kDa HSP70B–CDΔ2 complex may arise from a specific interaction but also from the recognition of CDΔ2 by HSP70B as a substrate due to misfolding of CDΔ2 induced by the absence of its J-domain. Compared with the weak HSP70B–CDΔ2 complex, the strong ~95-kDa 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 polymerization 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 complexes from soluble Chlamydomonas extracts by size by using BN-PAGE and transferred the complexes directly to nitro-cellulose membranes. Immunodetection with the CDJ2 antibody revealed a strong signal <66 kDa from monomeric or dimeric CDJ2 and weaker signals from complexes of ~150 and ~300 kDa (). 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.
Figure 8. Analysis of CDJ2 and VIPP1 complexes by BN-PAGE. Chlamydomonas total soluble proteins (Sol) were separated on a 6–15% native gel (BN-PAGE) and transferred directly to nitrocellulose (top two gels) or separated in the second dimension on a 10% (more ...)
CDJ2 in the second dimension was found mostly below 66 kDa and in a complex of ~150 kDa (). 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 absent 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 , , and ). 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 (). 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
). Exactly the same pattern of CDJ2- and VIPP1-containing complexes was detected when cells were lysed more gently by vortexing with glass beads instead of sonication (our unpublished data). This argues against the possibility of a nonspecific 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 unpublished data). This indicates that VIPP1 alone has the capability to form large oligomers and thus that the VIPP1 signal at >>669 kDa is not just low-molecular-weight VIPP1 associated with membrane vesicles, which may still be present in the soluble cell extracts and would hardly enter the native gel.
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.