Identification of a Hsp70 recognition domain within the rubisco small subunit transit peptide.
The interaction between SStp, the transit peptide of the precursor protein to the small subunit of Rubisco (prSSU) and two Hsp70 molecular chaperones, Escherichia coli DnaK and pea (Pisum sativum) CSS1, was investigated in detail. Two statistical analyses were developed and used to investigate and predict regions of SStp recognized by DnaK. Both algorithms suggested that DnaK would have high affinity for the N terminus of SStp, moderate affinity for the central region, and low affinity for the C terminus. Furthermore, both algorithms predicted this affinity pattern for >75% of the transit peptides analyzed in the chloroplast transit peptide (CHLPEP) database. In vitro association between SStp and these Hsp70s was confirmed by three independent assays: limited trypsin resistance, ATPase stimulation, and native gel shift. Finally, synthetic peptides scanning the length of SStp and C-terminal deletion mutants of SStp were used to experimentally map the region of greatest DnaK affinity to the N terminus. CSS1 displayed a similar affinity for the N terminus of SStp. The major stromal Hsp70s affinity for the N terminus of SStp and other transit peptides supports a molecular motor model in which the chaperone functions as an ATP-dependent translocase, committing chloroplast precursor proteins to unidirectional movement across the envelope.
Ivey
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RA
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee at Knoxville, Knoxville, Tennessee 37917, USA.
Subramanian
C
C
Bruce
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BD
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000465:Algorithms; D000595:Amino Acid Sequence; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry*
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; D012273:Ribulose-Bisphosphate Carboxylase