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. 2005 Mar;16(3):1165-77.
doi: 10.1091/mbc.e04-08-0736. Epub 2005 Jan 5.

J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1

Cuimin Liu  1 , Felix Willmund, Julian P Whitelegge, Susan Hawat, Bettina Knapp, Mukesh Lodha, Michael Schroda
Affiliations

J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1

Cuimin Liu et al. Mol Biol Cell. 2005 Mar.

Abstract

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 approximately 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 identified 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, approximately 150, and perhaps approximately 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.

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Figures

Figure 6.
Figure 6.
ATP dependence of the VIPP1–CDJ2–HSP70B interaction. 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.
Figure 5.
Figure 5.
Immunoprecipitation of CDJ2, VIPP1, and HSP70B. (A) Chlamydomonas soluble extract from ∼1.5 × 1010 cells was incubated with protein A-Sepharose coupled to antibodies of either preimmune (Pre), anti-CDJ2, or anti-VIPP1 serum, and membranes solubilized 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 nonreducing 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.
Figure 3.
Figure 3.
Immunoprecipitation of CDJ2. Six milliliters of a soluble extract from ∼1010 Chlamydomonas cells were incubated with protein A-Sepharose coupled to antibodies of either preimmune serum (Pre) or anti-CDJ2 serum. Precipitated proteins were eluted under reducing 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 nitrocellulose, and immunodecorated with anti-CDJ2 antibodies (right gel). The positions of IgG heavy chains (HC) and light chains (LC) are indicated.
Figure 1.
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, VIPP1, and the Chlamydomonas β-like protein 2 (cβlp2). The constitutively expressed cβlp2 served as loading control. (B) CDJ2 and VIPP1 protein accumulation 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 μg/lane). Constitutively 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 μg of total RNA per lane were hybridized with the same probes used in A.
Figure 2.
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 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).
Figure 4.
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 cells (WC, 14 μg) and fractions (S, 4 μg; M, 10 μg) 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 μg each) were separated on a 7.5–15% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunodecorated with antibodies against HSP70B, Cytf, CDJ2, VIPP1, CGE1, and mitochondrial carbonic anhydrase (CA).
Figure 7.
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 tags) were separated on a 7.5–15% SDS-polyacrylamide gel and visualized by silver staining. (B) CDJ2, CDΔ2, and HSP70B (2.6 μM each) were cross-linked, separated on 4–18% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunodecorated with antibodies against CDJ2 or HSP70B.
Figure 8.
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% 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.
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