Identification of an uncleavable targeting signal in the 70-kilodalton spinach chloroplast outer envelope membrane protein.
A cDNA clone encoding a cognate 70-kDa heat shock protein from the spinach chloroplast outer envelope (SCE70) was recently characterized (Ko, K., Bornemisza, O., Kourtz, L., Ko, Z. W., Plaxton, W. C., and Cashmore, A. R. (1992) J. Biol. Chem. 267, 2986-2993). Initial studies revealed that SCE70 is targeted to the chloroplast outer envelope membrane without further processing. To determine whether SCE70 possesses a "targeting domain," we tested the targeting ability of SCE70 proteins with various carboxyl- and amino-terminal deletions. Carboxyl-terminal deletions of up to 60% of the protein had no apparent effect on the targeting ability of SCE70. Amino-terminal deletions abolished targeting to the chloroplast except when the extreme NH2-terminal 48-amino acid sequence was retained. We further assessed the chloroplast-targeting ability of the NH2-terminal 48 amino acids by fusing to the foreign protein, mouse dihydrofolate reductase (DHFR). The resulting fusion protein, SCE70-DHFR, was localized to the outer envelope membrane of isolated chloroplasts. SCE70-DHFR exhibited targeting characteristics similar to native SCE70. The targeting of SCE70-DHFR was inhibited effectively by anti-SCE70 antibodies. Immunoprecipitation and chemical cross-linking experiments revealed that SCE70-DHFR is targeted to the same complex as SCE70 in the chloroplast envelope. These results suggest that the extreme NH2 terminus of SCE70 is required for directing SCE70 to a destination in the chloroplast outer envelope membrane, possibly through assembling the polypeptide into a protein complex.
Wu
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Department of Biology, Queen's University, Kingston, Ontario, Canada.
Ko
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K
D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D010944:Plants / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D003432:Cross-Linking Reagents; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase