FUNCTION AND REGULATION OF THE HEAT-SHOCK RESPONSE 
Susan L. Lindquist, Ph.D., Investigator 
When cells of all types are exposed to mildly ele- 
vated temperatures, ethanol, anoxia, heavy metal 
ions, or a wide variety of other stresses, they respond 
by producing a small number of proteins called the 
heat-shock proteins (HSPs) . This response is one of 
the most highly conserved genetic regulatory sys- 
tems known. Dr. Lindquist's research program fo- 
cuses on 1) investigation of the functions of the 
HSPs in protecting cells from the toxic effects of 
stress and in normal growth and development and 
2) use of the response as a model system to investi- 
gate mechanisms of genetic regulation, particularly 
post-transcriptional regulation. 
HSP Function 
Studies of the molecular functions of HSPs have 
concentrated on the yeast Saccharomyces cerevi- 
siae, because of the genetic methods of analysis pos- 
sible in this organism. This year Dr. Lindquist and 
her colleagues have concentrated on the analysis of 
hsp90 and hsplOO, the two largest HSPs in yeast. 
Yeast cells encode two closely related proteins in 
the hsp90 family, hsp82 and hsc82. Disruption of 
the gene encoding either protein prevents growth at 
high temperatures, while deletion of both is lethal. 
Thus hsp90 is a vital protein that is required at 
higher concentrations for growth at higher tempera- 
tures. The mammalian hsp90 gene can compensate 
for the loss of both yeast genes, demonstrating that 
the functions of these proteins have been highly 
conserved. This is important, because the mamma- 
lian protein has been characterized extensively at 
the biochemical level, and Dr. Lindquist's labora- 
tory wishes to take advantage of yeast genetic meth- 
ods to investigate such functions in vivo. In collabo- 
ration with Dr. Keith Yamamoto, Dr. Lindquist's 
laboratory demonstrated that hsp90 is required for 
the formation of functional steroid hormone recep- 
tors. This proved that the interaction between 
hsp82 and steroid receptors is important in vivo. It 
also caused a substantial revision in thinking about 
hsp90 functions, since hsp90 had previously been 
characterized as a repressor of receptor function. 
This year Drs. Yamamoto and Lindquist explored 
the interaction between hsp90 and p60^ As with 
the steroid receptors, the results indicate that hsp90 
is essential for the formation of active kinase. More- 
over, the expression of src kinase in yeast cells 
causes a cell cycle arrest. This indicates that src has 
specific targets in yeast cells. Because of the conser- 
vation of cell cycle regulation, the identification of 
this target may be relevant to vertebrate oncology. 
This work is being supported by grants from the Na- 
tional Institutes of Health and the Human Frontiers 
in Science Program. 
This past year Dr. Lindquist's laboratory has in- 
creasingly focused attention on hsplOO. Cells carry- 
ing mutations in the gene encoding this protein 
grow normally at both high and low temperatures, 
but are 100- to 1 , 000-fold more sensitive than wild- 
type cells to heat killing at 50°C and to ethanol kill- 
ing at concentrations of 20%. The protein is very 
highly conserved, and heat-inducible members of 
the family are found in bacteria and mammals. Sur- 
prisingly, they are not found in Drosophila. This 
year the laboratory discovered that Drosophila cells 
make a heat-inducible transcript that is homologous 
to hsplOO, indicating that expression of the protein 
was lost recently in the evolution of these flies. Dr. 
Lindquist and her colleagues are currently trying to 
determine how widespread this loss is among in- 
sects and if the loss of the protein might confer a 
growth advantage that would compensate for the 
presumed disadvantage in thermotolerance. The 
hsplOO proteins have two nucleotide-binding sites, 
and both are required for thermotolerance . The puri- 
fied protein is an ATPase and assembles into a hex- 
amer in the presence of ATP. Currently the labora- 
tory is attempting to determine what substrates 
hsplOO interacts with and what effects mutations in 
the nucleotide-binding sites have on substrate inter- 
actions and self-assembly. To define proteins that 
might interact with hspl04, the laboratory has initi- 
ated a genetic search for dominant negative muta- 
tions in hspl04. Several have been produced and 
are now being characterized. Finally, mutations are 
being produced in another member of the hsplOO 
family in yeast, in collaboration with Dr. Chris 
Davies (University of North Carolina) . 
This year Dr. Lindquist and her colleagues have 
expanded a new line of research aimed at determin- 
ing the function of HSPs in protecting animals from 
the developmental anomalies that are caused by 
high temperatures. They have developed a new sys- 
tem for site-directed recombination in Drosophila 
and are using it to create isogenic flies carrying 
various copy numbers of HSP genes and gene muta- 
tions. Flies carrying 10 extra copies of the hsp70 
gene survive heat shocks better than wild-type flies, 
at least in the early embryonic stages, indicating that 
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