Interaction Mating

Interaction mating allows the testing of a large number of protein-protem interactions for predefined group of proteins (10). In this method, a series of DBD-P(n) and AD-P(x) fusions are transformed independently into hap-loid yeast strains of opposite mating types, and cross-gridded together to select diploids. Combinations containing interacting fusion proteins can be rapidly detected by replica of diploids to X-gal and leu- selective plates (see Fig. 3).

1. Grow cultures of EG Y48 and of RF Y206 for transformation (see above for transformation protocols)

Into EGY48 (MATa), transform the following combinations of plasmids p-202-Pl +JK103 plate to ura-his-

Into RFY206 (MATa), transform the following plasmids:

pJG4-5-Pl plate to trp-

pJG4-5 plate to trp-

Incubate transformation plates at 30°C for 2—3 d until colonies appear

ura-hfs-glucose trp- glucose

EGY48, MAT« RFY206, MATa ura-hfs-glucose trp- glucose

ura-his-trp-XGal glucose ura-his-trp-XGal galactose ura-his-trp-XGal glucose ura-his-trp-XGal galactose

2 Make ura-his- and trp- master plates (as appropriate) containing at least two picked colonies for each transformation

3 On a ura-his- plate, use a toothpick to make parallel streaks of EGY48 yeast containing p-202-P(n) and JK103. eight to ten separate colonies can conveniently be streaked on a single plate (see Fig 3).

On a trp- plate, use toothpicks to make parallel streaks of RFY206 containing a series of JG4-5-fused proteins

It is helpful to include the cross combination of p-202 and pJG4-5 on each plate, as negative control

4. Replica plate these two sets of streaks onto YPD plates, where cells of different mating types can mate and form diploids at the intersections of the horizontal and vertical lines (Fig 3) Note, it is important to use a separate velvet for each replica to avoid cross-contamination. Incubate plates overnight at 30°C

5. Using the YPD plates containing the mated yeast as master plates, replica plate to a ura-his-trp- glucose plate. Incubate plates overnight to two d at 30°C This will allow outgrowth of diploid yeast containing all DBD- and AD-fused proteins, and reporters Note, some researchers omit this step, and proceed directly from the YPD plate to selective media In our hands, we have found that including this intermediate step to first select for diploids leads to clearer results

6. Using the ura-his-trp- plate as a master, replica plate to the four following combinations of plates a. ura-his-trp- glu XGAL.

b. ura-his-trp-gal/raf XGAL c ura-his-trp-leu- glu d ura-his-trp-leu- gal/raf Incubate plates 2-3 d at 30°C

7. If two proteins interact specifically, then for plates containing galactose/raffi-nose (inducing the expression of the AD-fusion) that DBD/AD-fused combination of proteins will cause enhanced growth on ura-trp-his-leu- medium and will cause enhanced blue color on plates containing X-gal, relative to background (defined as the DBD-fusion coexpressed with JG4-5 vector) These same DBD/AD

Fig. 3. (previous page) Mating panel assay is illustrated In separate transformations, MATa EGY48 yeast are transformed with a series of p-202 fusions and a LacZ reporter plasmid, whereas MATa RFY206 are transformed with a series of AD-fusions Transformants are streaked as parallel lines on a ura-his- (EGY48) or trp- (RFY206) plate. A replica block is used to cross grid the EGY48 and RFY206 plates onto a YPD plate, where mating occurs After 12 h, cross gridded colonies are optionally replica plated onto ura-his-trp- glucose to select for diploids In a final replica plating, colonies are lifted from the ura-his-trp- glucose plate (or the YPD plate) to plates selective for LacZ and LEU2 expression (see text). Growth specific to galactose leumedium and blue color specific to galactose X-gal medium indicate positive interactions Note, any LexA-fusion with residual ability to activate transcription will also be positive on glucose media: one example is shown.

combinations will show no enhancement of growth or blue color on plates containing glucose as a sugar source 8 Some AD-fused proteins enhance transcription relatively nonspecifically with a large number of other proteins (HSP70 is a good example, as is the fragment of bicoid encoded in RFHMI) These are likely to show a random pattern of interaction with multiple p-202 fused proteins in a mating grid, the significance of which is not known

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