Protein Folding In Vivo 
Arthur Harwich, M.D. — Associate Investigator 
Dr. Harwich is also Associate Professor of Human Genetics and Pediatrics at Yale University School of 
Medicine. He received A.B. and M.D. degrees in biomedical sciences from Brown University. His internship 
and residency training in pediatrics were done at Yale. His postdoctoral research training was at the Salk 
Institute with Walter Eckhart and at Yale University with Icon Rosenberg. 
WE have been using mitochondria, the intra- 
cellular organelles that carry out energy 
metabolism, as a system for investigating how 
proteins cross biological membranes. 
Most of the proteins of mitochondria are first 
made outside the organelles, in the cytosol, and 
then imported through both an outer and inner 
mitochondrial membrane to reach the innermost 
"matrix" compartment. In order to traverse the 
membranes, the newly made proteins are first un- 
folded on the cytosolic side. After import, they 
refold on the inside of the organelle into their 
biologically active conformations. 
It has always been assumed that this process of 
refolding is a spontaneous event, much like the 
refolding of many denatured proteins observable 
in a test tube. Yet we have identified a mutant cell 
in which mitochondrial proteins were imported 
into the matrix but failed to fold into biologically 
active forms. The mutation was found to affect a 
protein that normally resides in the matrix, called 
heat-shock protein 60 (hsp60). 
This protein was originally identified by the 
observation that its level was increased about 
twofold in response to incubation of cells at high 
temperatures. It is abundant, however, even be- 
fore heat shock, and our genetic analysis demon- 
strated that, consistent with a critical baseline 
function, hsp60 is required not only at high tem- 
peratures but at all temperatures. The increased 
level produced in response to heat stress could 
represent an effort of the cell to efficiently refold 
mitochondrial proteins that heat has denatured. 
In the mitochondrial matrix, hsp60 is found in 
a higher order structure, a complex. Fourteen 
copies of the protein are arranged in two stacked 
rings, a "double donut." Each ring contains seven 
radially arranged copies of hsp60. How does this 
complex function? Our studies have demon- 
strated that unfolded mitochondrial proteins en- 
tering the matrix space become associated with 
the surface of the hsp60 complex. Then, in steps 
requiring both energy and a second protein com- 
ponent, the polypeptides are folded into their ac- 
tive forms and released from the complex. 
We are now trying to dissect the mechanism of 
hsp60-directed folding. We believe that the path- 
way of folding must be dictated by the amino acid 
sequence of the "substrate" protein to be folded, 
not by the hsp60 complex, because we have used 
the complex to fold proteins that normally reside 
outside mitochondria. 
It seems that hsp60 acts by speeding up, or 
"catalyzing," the folding of proteins. How does it 
do this? One possibility is that it simply prevents 
domains of proteins from wrongfully interacting, 
either with each other or with nearby proteins in 
the mitochondrial matrix, a "chaperone" func- 
tion. Another possibility is that the complex ac- 
tively promotes the progression of an unfolded 
protein through a series of folding steps. Because 
we can now reconstitute hsp60-mediated folding 
in a test tube, we can use biochemical and bio- 
physical techniques to examine the folding mech- 
anism more precisely. 
How general is the utilization of folding ma- 
chinery in living cells? Because mitochondria ap- 
parently arose from bacteria (one cell ingested 
another), it is not surprising that a structurally 
related component has been found in bacteria. 
Here a function like that of hsp60 is implied by 
an experiment carried out many years ago. When 
Escherichia coli cells partially defective in the 
hsp60-related protein were infected with virus, 
the newly made virus coat proteins could not as- 
semble to make new virus particles. We surmise 
that the hsp60-related component is likely to be 
used not only to fold and assemble proteins of 
infecting viruses, but also to fold most if not all 
newly synthesized proteins indigenous to the bac- 
terial cell. We are currently testing whether this 
is the case by producing a severe defect in the 
hsp60-related component. 
Like bacteria, cells of higher organisms might 
utilize a "machine" to fold newly made proteins 
into their active forms. We are currently taking 
both genetic and biochemical approaches toward 
identifying such a component. 
How does a folding machine like the hsp60 
complex itself get assembled? It seemed possible 
that while all mitochondrial proteins tested to 
date utilize hsp60 for their folding and assembly. 
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