J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1.
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.
Liu
Cuimin
C
Plant Biochemistry, Institute of Biology II, University of Freiburg, D-79104 Freiburg, Germany.
Willmund
Felix
F
Whitelegge
Julian P
JP
Hawat
Susan
S
Knapp
Bettina
B
Lodha
Mukesh
M
Schroda
Michael
M
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D016825:Chlamydomonas reinhardtii; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D030541:Databases, Genetic; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D006358:Hot Temperature; D047468:Immunoprecipitation; D008027:Light; D046911:Macromolecular Substances / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000378:metabolism; D018087:Plastids / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013696:Temperature; D020524:Thylakoids / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D018840:HSP70 Heat-Shock Proteins; C491129:HSPA7 protein, human; D046911:Macromolecular Substances; D008565:Membrane Proteins; D018832:Molecular Chaperones; C424449:VIPP1 protein, Synechocystis; D049452:Green Fluorescent Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D005976:Glutaral