Characteristics of an Hsp70 homolog localized in higher plant chloroplasts that is similar to DnaK, the Hsp70 of prokaryotes.
Members of the 70-kD heat-shock protein (Hsp70) family are important cellular factors that are thought to mediate protein folding and assembly. A chloroplast-localized Hsp70 homolog (Chsp70) was recently identified based on its similarity to DnaK, the Hsp70 homolog of Escherichia coli (D. Amir-Shapira, T. Leustek, B. Dalie, H. Weissbach, N. Brot [1990] Proc Natl Acad Sci USA 87: 1749-1752). To learn more about the function of Chsp70, we purified the protein from Spinacia oleracea chloroplasts by ATP-agarose affinity chromatography. A single, 75,000-D protein was isolated which becomes phosphorylated on a threonine residue when incubated with [gamma-32P]ATP and 10 mM Ca2+, a property similar to DnaK. Chloroplast fractionation and immunoblot analysis showed that Chsp70 is a soluble stromal protein. Chsp70-specific antiserum was used to clone a partial cDNA that shows greater homology with Hsp70 from prokaryotes than with cytoplasmic Hsp70 from eukaryotes. The antiserum and cDNA were used to study Chsp70 expression. Following heat shock of spinach seedlings at 37 degrees C, Chsp70 synthesis increase 12-fold, the level of Chsp70 mRNA increases 5-fold, and the level of Chsp70 protein increases less than 2-fold. Chsp70 is constitutively expressed in all spinach tissues, indicating that it is likely to be localized in all plastid types. The highest levels occur in seeds, leaves, florets, and seedlings grown in the light. Lower levels occur in roots, stems, and etiolated seedlings.
Wang
H
H
Center for Agricultural Molecular Biology, Rutgers University, New Brunswick, New Jersey 08903.
Goffreda
M
M
Leustek
T
T
D001426:Bacterial Proteins / Q000096:biosynthesis; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007106:Immune Sera; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000096:biosynthesis; D010944:Plants / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007106:Immune Sera; D010940:Plant Proteins; D012333:RNA, Messenger; C063842:dnaK protein, E coli