The
BIG Picture
Research
in our laboratory is aimed at understanding the roles of heat
shock proteins (Hsps) and other factors that affect heat stress tolerance
in plants. Hsps are expressed by virtually all cells under conditions of
high temperature and many other stresses.
Hsps are believed
to protect cells against permanent heat injury and to confer increased
thermotolerance to cells. Most Hsps function as molecular chaperones, which
are a diverse group of proteins that share the property of binding to other
proteins that are in unstable structural states. Chaperones facilitate
a range of processes including protein folding, transport of proteins across
membranes, modulation of protein activity, regulation of protein degradation,
and prevention of irreversible protein aggregation. The latter activity
is believed to be critical to survival of high temperature stress.
Our research
is focused primarily on Hsps from higher plants, including the model organism
Arabidopsis
thaliana, but we also work with single-celled organisms for genetic
studies, including the cyanobacterium Synechocystis PCC 6803. We
use a range of experimental methods including molecular biology, biochemistry,
classical genetics and generation of transgenic plants.
The
small Hsps
A major
focus of the lab is structure and function of the small Hsps (sHsps).
The Hsp/a-crystallin
family of proteins are conserved
in both eukaryotes and prokaryotes, and they are produced at significant
levels in cells
experiencing heat stress, supporting
the hypothesis that they have an ancient and conserved role in survival
of high temperature stress.
Negative stain electron microgrpah of pea Hsp18.1 complexes.
Unique
evolution of the plant sHsps
The sHsps
appear to represent a special adaptive mechanism of plants to high temperature,
as plants have evolved sHsps that are targeted to not only the cytoplasm,
but also to the chloroplast, mitochondrion and endoplasmic reticulum. Evolutionary
analysis shows that these sHsp
families are unique to the plant lineage, and that they arose before
the Bryophytes, 400 million years ago. The chloroplast and mitochondrial
proteins represent a unique type of organelle protein evolution, probably
arising from a nuclear gene that duplicated and was selected on for function
in the organelle. This work was done primarily by Liz Waters, with help
from lots of other folks who had cloned genes in the past (Thanks especailly
to Binh Chau).
sHsps
in big packages - the "holey soccer ball structure"
Although
the sHsp monomeric molecular mass ranges from 15-40 kDa, in their native
state they are found as oligomers consisting of 9-32 subunits, depending
on the species. The sHsps are overall diverse in sequence, but share a
characteristic ~100 residue, C-terminal domain (the "a-crystallin
or heat shock domain") with signature amino acid motifs and similarity
of predicted secondary structure. Despite the ubiquitous nature of these
proteins, the only available 3-D structural data comes from the recent
solution of the a-crystallin domain (residues 33-147) of Methanococcus
jannaschii Hsp16.6 (45).
MjHsp16.6
is a symmetrical 24 subunit oligomer appearing as a hollow sphere with
an outer diameter of 12 nm.
We have
initiated X-ray crystallographic studies of plant small Hsp structure with
Dr. Christine Slingsby's lab at Birkbeck College, University of London.
We hope to complete the 3-D structure of an sHsp in the year 2000. This
work has benefitted from many years of biochemistry by Gary Lee, and more
recently by Eman Basha.
Keeping
things afloat - sHsps as molecular chaperones
Although
how sHsps might protect plants from the stress effects of high temperature
remains unknown, like other Hsps, they have been proposed to have molecular
chaperone activity. We have proposed a model in which sHsps protect
other proteins from irreversible heat denaturation and facilitate reactivation
of denatured proteins. Using purified proteins we can show that sHsps prevent
proteins from aggregating and dropping out of solution when heated.
Model substrates that are denatured and bound to sHsps can then be refolded
by other chaperones - Hsp70 and cofactors DnaJ (and grpE in prokaryotes).
We have followed this entire reaction in a test tube, thanks to the efforts
of Gary Lee, which were followed up on by Dave Kim, Adam Geach and now
by Kenny Friedrich.
Heat-denatured proteins can either aggregate to form insoluble complexes, or can be prevented from aggregating by binding to sHsps. The denatured protein can then be degraded by proteases in the cell, or refolded by ATP-dependent chaperones like HSP70.
Genetics
in pond scum - Synechocystis as a model organism for sHsp structure
and function
To get
at sHsp function in vivo, we have begun studies with Synechocystis
sp. PCC6803. This single-celled cyanobacterium can be easily transformed
and the entire genome sequence of ~3,200 genes is known. Synechocystis
has a single sHsp gene, Hsp16.6, and deletion of this gene leads to a defect
in growth at high temperature. We are now using
Synechocystis for
structure and function studies by making mutations in the Hsp16.6 gene
and testing their phenotype. We are also interested in defining the proteins
that may be protected by Hsp16.6 in vivo. We have found that a specific
subset of Synechocystis proteins are bound to Hsp16.6 following
heat stress in vivo. The bound proteins can be released in the presence
of other chaperones in an ATP-dependent fashion, suggesting they represent
true in vivo substrates. This work was pioneered by Kim Giese and Gary
Lee and is also being worked on by Nicole Buan and Eman Basha.
More than just heat stress - sHsps are important in seed development
Seed development in Arabidopsis thaliana. The timing and relative expression levels of various proteins, including sHsps, is indicated.
Seed development is also stressful - seeds dry during maturation to less than 10% of total water content and then rehydrate in a matter of hours during germination. sHsps seem to play a role in late seed maturation processes, possibly involved in dormancy, desiccation tolerance and seed viability. Nadi Wehmeyer did a terrific job investigating the expression of sHsps and looking at the effects of antisense suppression on seed phenotype. Results of the latter studies are not ready to be revealed!
Expression of sHsp in Arabidopsis seeds. On the left, sHsps are present in all but the tip of the cotyledons and the tip of the radicle. A control seed is shown to the right.
From small to big - the Hsp100/ClpB
proteins
We also
study the Hsp100 family of proteins. These proteins are believed to pry
apart protein aggregates formed by heat stress so that they can be refolded
by other chaperones. Like the sHsps, in addition to being expressed during
heat stress, they are also regulated during seed development. Antisense
plants were made in collaboration with Christine Queitsch in Susan Lindquist's
lab at the University of Chicago. These results will be posted soon!!!
The HSP100 proteins bind to aggregates of denatured proteins and cause them to dissociate from each other. The previously aggregated polypeptides are then refolded with the help of DnaJ and HSP70.
The awesome power of Arabidopsis
genetics
Using mutagenized
Arabidopsis
plants we are working towards demonstrating the requirement of specific
Hsps in thermotolerance and also toward isolating mutants defective in
the acquisition of thermotolerance. We now have mutants of Arabidopsis
that fail to adapt to high temperatures like wild type. We know that one
of the mutations is in a gene encoding an Hsp. Stay-tuned to learn more
about this project, which has been spearheaded by Suk-Whan Hong and a fleet
of dedicated Arabidopsis-screening undergrads.
Arabidopsis plants which are pretreated with a mild heat stress develop thermotolerance and are more resistant to heat damage than plants which recieve no pretreatment.
We acknowledge current grant support from the USDA and DOE:
USDA NRICGP 99-351007618
"Plant
Thermotolerance: The role of small heat shock proteins and other factors"
DOE - Energy Biosciences Program
"Cytosolic
HSP100 Proteins and Stress Tolerance in Plants"
We also
thank the USDA for over 10 years of continuous support, the American Cancer
Society and the NIH for multiple years of support.
Arabidopsis thaliana (Columbia
wild-type).
