J-Domain Protein CDJ2 and HSP70B Are a Plastidic Chaperone Pair That Interacts with Vesicle-Inducing Protein in Plastids 1

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 ϳ28-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 Ͼ Ͼ669, ϳ150, and perhaps ϳ300 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. INTRODUCTIONChaperones 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 Nterminal ATPase domain and a C-terminal substrate-binding domain. ATP hydrolysis at the ATPase domain regulates substrate binding and release. Substrate proteins recognized by Hsp70 expose hydrophobic regions, a characteristic feature not only of nonnative proteins, but also of native Hsp70 substrates. Binding of Hsp70 to hydrophobic regions prevents 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 response (Tomoyasu et al., 1998), the uncoating of clathrincoated 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 represent an important class. J-domain cochaperones contain a highly conserved J-domain that is responsibl...

Arabidopsis Cotyledon-Specific Chloroplast Biogenesis Factor CYO1 Is a Protein Disulfide Isomerase

“…DnaJ proteins are molecular chaperones that specifically regulate DnaK-like proteins involved in protein folding. Hsp70s, DnaK homologs in chloroplasts, play an important role in a number of processes (Liu et al, 2005;Lu et al, 2006). However, CYO1 lacks the J domain responsible for stimulating DnaK ATPase activity.…”

“…Maize BSD2 also has a Cys-rich zinc binding domain similar to that in DnaJ and is targeted to chloroplasts (Brutnell et al, 1999). BSD2 is not involved in general photosynthetic complex assembly or protein import but is required for the posttranslational regulation of RBC-L. CDJ2, a chloroplast DnaJ homolog, may function during the biogenesis and/or maintenance of thylakoid membranes (Liu et al, 2005). This protein has the J domain that interacts with Hsp70 but lacks a Cys-rich domain.…”

“…BSD2 is required for posttranslational regulation of the L subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) (Brutnell et al, 1999). Chloroplast DnaJ homolog2 (CDJ2) is required for the biogenesis and/or maintenance of thylakoid membranes (Liu et al, 2005). RB60 is an atypical PDI that functions as a member of a redox regulatory protein complex controlling translation in Chlamydomonas reinhardtii chloroplasts (Kim and Mayfield, 1997).…”

Glycerolipid transfer for the building of membranes in plant cells

“…Recent data demonstrate that VIPP1 organizes in a high molecular mass complex closely associated with the inner envelope membrane and suggest that the C-terminus of the protein protrudes from the complex into the stroma of chloroplasts possibly for interaction with some other proteins [77]. Accordingly, soluble VIPP1 interacts with a HSP70B/CDJ2 chaperone pair [78]. By analogy with the action of the auxilin/Hsc70 chaperone pair with clathrin on clathrin-coated 20 vesicles, HSP70B/CDJ2 might disassemble and/or assemble VIPP1 oligomers to recycle the system for another turn of vesicle formation/transport [78].…”

“…Accordingly, soluble VIPP1 interacts with a HSP70B/CDJ2 chaperone pair [78]. By analogy with the action of the auxilin/Hsc70 chaperone pair with clathrin on clathrin-coated 20 vesicles, HSP70B/CDJ2 might disassemble and/or assemble VIPP1 oligomers to recycle the system for another turn of vesicle formation/transport [78].…”