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Plant Physiol, April 2000, Vol. 122, pp. 1289-1300
Identification of a Hsp70 Recognition Domain within the Rubisco
Small Subunit Transit Peptide1
Robert A.
Ivey III,
Chitra
Subramanian, and
Barry D.
Bruce*
Department of Biochemistry and Cellular and Molecular Biology
(R.A.I., B.D.B.), The Graduate Group in Plant Physiology and Genetics
(C.S., B.D.B.), Center for Legume Research (B.D.B.), University of
Tennessee at Knoxville, Knoxville, Tennessee 37917
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.
1
This work was supported by the Cell Biology
Program at the National Science Foundation (grant nos. MCB-9401840 and
MCB-9604535 to B.D.B.) and by The Science Alliance Program at the
University of Tennessee-Knoxville.
*
Corresponding author; e-mail bbruce{at}utk.edu; fax 423-974-6306.
© 2000 American Society of Plant Physiologists
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