Plant Molecular Biology 39: 871–881, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Molecular characterization of a plant mitochondrial chaperone GrpE
, Vijay S. Reddy
, Roger N. Beachy
and Claude M. Fauquet
International Laboratory for Tropical Agricultural Biotechnology (ILTAB/ORSTOM-TSRI), Division of Plant
Biology-BCC206, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
author for correspondence);
Department of Molecular Biology-MB31, The Scripps Research Institute, 10550
North Torrey Pines Road, La Jolla, CA 92037, USA.
Received 4 August 1998; accepted in revised form 22 October 1998
Key words: Arabidopsis thaliana, chaperone, GrpE structure, mitochondrial GrpE, mitochondrial transit peptide,
Escherichia coli DnaK (Hsp70) cooperates with DnaJ and GrpE in its essential role as a molecular chaperone.
Function of mitochondrial Hsp70 (mHsp70) in protein folding and organellar import in eukaryotes is critically
dependent on GrpE. We cloned two genes from tobacco (Nicotiana tabacum) BY2 cells based on peptide sequences
from a puriﬁed protein. The predicted amino acid sequences of both clones resembled that of GrpE from E. coli
and its homologues from eukaryotes, and a cDNA clone from Arabidopsis thaliana. One gene (Type 1) encoded a
deduced protein that was identical to the puriﬁed protein while the other (Type 2) encoded a deduced protein that
has 80% sequence identity to Type 1. Both tobacco and Arabidopsis thaliana GrpE homologues bound to DnaK
and ATP inhibited this binding. The tobacco GrpE homologue contained a typical N-terminal mitochondrial target
presequence of 64 residues and the presequence directed the green ﬂuorescent protein to tobacco mitochondria.
The tobacco GrpE homologue also associated with mHsp70 when reintroduced into BY2 protoplasts, and this
association was disrupted by ATP. A three-dimensional structure for the tobacco GrpE homologue was modeled
based on the X-ray structure of E. coli GrpE complexed with DnaK. The modeled structure has the same overall
structure as E. coli GrpE. We propose that the tobacco GrpE homologue interacts with mHsp70 in a manner
analogous to E. coli GrpE with DnaK and designate it as tobacco mitochondrial GrpE (NtmGrpE).
Heat shock proteins (Hsp) are ubiquitous and have
multiple functions, including protein folding and
translocation across membranes. Among the heat
shock proteins, the Hsp70 family of proteins are best
studied; these proteins act by binding and releasing
extended peptide segments enriched in hydrophobic
side chains in an ATP-dependent manner [3, 26, 30,
35]. Hsp70 homologues in eukaryotes are found in
the cytoplasm, nucleus, ER/Golgi, plastids, and mi-
tochondria [7, 10, 12, 25, 29]. The Hsp70 homologues
found in mitochondria and chloroplasts are similar to
The nucleotide sequence data reported will appear in the Gen-
Bank Nucleotide Sequence Database under the accession numbers
AF098635 and AP098636.
E. coli DnaK (Hsp70) in both sequence and func-
tion. The function of DnaK as molecular chaperone
is enhanced more than 50-fold by association with
the chaperone DnaJ and the nucleotide exchange fac-
tor GrpE [32, 35, 44]. ATP-bound DnaK binds and
releases peptides in a rapid manner while the ADP-
bound DnaK binds peptides in a stable manner. DnaJ
stimulates the ATP hydrolysis activity of DnaK and
peptide release is catalyzed by GrpE. Recent stud-
ies in yeast and mammals established the presence of
GrpE and DnaJ homologues in mitochondria. In yeast
and mammals, the function of mitochondrial Hsp70
(mHsp70), but not of the cytosolic Hsp70, critically
depends on GrpE and DnaJ homologues [2, 6, 11,
23, 28]. They facilitate protein folding and import into