tivity requires secondary, lower affinity interactions 
of fibrin with either of the two kringle domains. 
The binding of the t-PA molecule to specific recep- 
tors on hepatic cells also involves sequences within 
the finger and/or EGF-like domains. Finally, the ab- 
sence of heavy-chain domains has no effect on the 
interaction of PAI-1 with the catalytic light chain. 
Although the three-dimensional structure of t-PA 
has not been elucidated, Dr. Gething and her col- 
leagues have been able to model the EGF-like do- 
main, the kringle domains, and the light-chain/in- 
hibitor complex, using the known structures of 
homologous proteins. Site-directed mutants de- 
signed using these proposed structures have pro- 
vided information about the individual amino acid 
sequences that interact with the effector molecules. 
For example, the group has generated variant en- 
zymes that are efficient, fibrin-stimulated plasmino- 
gen activators but 1) are resistant to inhibition by a 
variety of serpins, including PAI-1, or 2) do not bind 
to the t-PA receptor(s) involved in clearance of the 
enzyme in the liver. Because these mutant enzymes 
should have an extended effective life in the circu- 
lation, they may have significant potential for use in 
thrombolytic therapy of patients with myocardial 
infarction. 
III. Endoplasmic Reticulum Proteins Involved in 
Folding and Mobilization of Nascent Polypeptides. 
Prefolded or malfolded forms of HA bind to a 77 
kDa cellular protein present in the lumen of the 
ER. A second ER protein (94 kDa) associated with 
nascent, unglycosylated HA synthesized in the pres- 
ence of drugs that inhibit the addition of amino- 
linked oligosaccharides to the newly synthesized 
polypeptide. As part of the effort to understand the 
role of protein folding in intracellular transport, 
PUBLICATIONS 
biochemical and genetic techniques are being used 
to characterize the structure and function of these 
two ER proteins. 
A full-length cDNA encoding the 77 kDa protein 
has been cloned and expressed from murine cells. 
On the basis of amino acid sequence, immunologi- 
cal reactivity, and functional activity, it has been es- 
tablished that this protein corresponds to two pre- 
viously described ER proteins, the immunoglobulin 
heavy-chain binding protein (BiP) and the glucose- 
regulated protein (GRP78), and that it is a member 
of the HSP70 multigene family, which includes the 
cytoplasmic 70 kDa heat-shock proteins. A full- 
length cDNA encoding the murine 94 kDa ER pro- 
tein has also been cloned. Like GRP78, GRP94 is re- 
lated to cytoplasmic heat-shock proteins, displaying 
45% sequence identity with HSP90. These glucose- 
regulated proteins, which are major constituents of 
the ER of mammalian cells, are synthesized consti- 
tutive ly under normal growth conditions but are in- 
duced under a variety of conditions of stress, with 
the common denominator the accumulation of un- 
folded polypeptides in the ER. 
The gene encoding BiP from the yeast Saccha- 
romyces cerevisiae has been cloned, using the mu- 
rine BiP cDNA as a hybridization probe. This gene 
is essential for viability of yeast cells. Surprisingly, 
the coding sequence of yeast BiP is identical to that 
of the KAR2 gene, one of a class of genes involved 
in nuclear fusion after mating of yeast cells. Expres- 
sion of mammalian BiP in S. cerevisiae can comple- 
ment a mutant allele of KAR2 that is temperature 
sensitive for growth and nonconditionally defective 
for karyogamy. 
Dr. Gething is also Professor of Biochemistry at 
the University of Texas Southwestern Medical Cen- 
ter at Dallas. 
Articles 
Boose, J.A., Kuismanen, E., Gerard, R., Sambrook, J., and Gething, M.J. 1989. The single-chain form of tissue- 
type plasminogen activator has catalytic activity: studies with a mutant enzyme that lacks the cleavage site. 
Biochemistry 28:635-643. 
Gallagher, R, Henneberry, J., Wilson, J., Sambrook, J., and Gething, M.J. 1988. Addition of carbohydrate side 
chains at novel sites on influenza virus hemagglutinin can modulate the folding, transport, and activity of 
the molecule. J Cell Biol 107:2059-2073. 
Gething, M.J., McCammon, K., and Sambrook, J. 1989. Protein folding and intracellular transport: evaluation 
of conformational changes in nascent exocytotic proteins. Methods Cell Biol 32:185-206. 
Gething, M.J. , and Sambrook, J. 1989. Protein folding and intracellular transport: studies on influenza virus 
hemagglutinin. Biochem Soc Trans 55:155-165. 
Continued 
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