Cloning in Saccharomyces cerevisiae and other fungi - Principles of Gene Manipulation - page 169

c-myc

Aga 2 HA MCS c-myc

Cloned
gene
product

(a) (b)

HA

Aga 2
s
s

Fig. 9.10 Yeast surface display of
heterologous proteins. (a) Schematic
representation of surface display.
(b) A vector used for facilitating surface
display. MCS, multiple cloning site;
HA, haemagglutinin epitope; c-myc,
c-myc epitope. See text for details.

s
s
Aga 1

Yeast cell wall

been inserted in the correct translational reading
frame, its gene product will be synthesized as a
fusion with the Aga2p subunit. The fusion product
will associate with the Aga1p subunit within the
secretory pathway and be exported to the cell sur-
face (Boder & Wittrup 1997).
In practice, the gene fusion is somewhat more
complicated. Usually the cloned gene product is
sandwiched between two simple epitopes to permit
quantitation of the number of fusion proteins per
cell and to determine the accessibility of different
domains of the fusion protein (Fig. 9.10).

DNA-binding domain hybrid

BAIT

DBD GAL4 (1-147)

UAS G GAL1-lacZ

Activation domain hybrids encoded by a library

TAD
GAL4 (768-881)

PREY

Y 1-n

Detecting protein-protein interactions

Chien et al . (1991) have made use of the properties of
the GAL4 protein to develop a method for detecting
protein-protein interactions. The GAL4 protein has
separate domains for the binding to UAS DNA and
for transcriptional activation. Plasmids were con-
structed which encode two hybrid proteins. The first
consisted of the DNA-binding domain (residues
1-147) of the GAL4 protein fused to a test protein.
The second consisted of residues 768 - 881 of the
GAL4 protein, representing the activation domain,
fused to protein sequences encoded by a library of
yeast genomic DNA fragments. Interaction between
the test protein (bait) and a protein encoded by one
of the library plasmids (prey) led to transcriptional
activation of a reporter gene (Fig. 9.11). This method
is known as the two-hybrid system. The two-hybrid
system has become a major tool in the study of
protein-protein and protein-ligand interactions (see

UAS G GAL1-lacZ

Interaction between DNA-binding domain
hybrid and a hybrid from the library

TAD
GAL4 (768-881)

BAIT PREY

Y i

DBD GAL4 (1-147)

UAS G GAL1-lacZ

Fig. 9.11 Strategy to detect interacting proteins using
the two-hybrid system. UAS G is the upstream activating
sequence for the yeast GAL genes, which binds the GAL4
protein, DBD is a DNA-binding domain and TAD is a
transcription-activation domain. Interaction is detected by
expression of a GAL1- lacZ gene fusion. (Reproduced courtesy
of Dr S. Fields and the National Academy of Sciences.)