strength of the interaction is normally higher when using a
LexA-based bait plasmid in comparison to the GBD-based
one and, in addition, there are good anti-LexA antibodies in
the market that can be used to test the production and quality
of the bait fusion protein (unfortunately no good commercial
antibody for Gal4-GBD is in the market yet).- There are alternative vectors in the market that produce LexA-
fusion proteins both with LexA at the N-terminus or as a
C-terminal fusion. The choice depends on the stability of the
fusion protein and on the preservation of the function of the
AMPK subunit. In our hands, N-terminal fusions of LexA give
better results than C-terminal fusion proteins. - The main difference between the available GAD-based plas-
mids is the type of multicloning site present in them. Both
pACT2 and pGADT7 contain an HA-epitope between the
GAD and the corresponding ORF which can be used to detect
the production and quality of the prey fusion proteins
(GAD-HA-prey fusion protein). AMPK subunits should be
also subcloned into these plasmids to confirm the interaction
with the putative substrate. In this way both directions of the
two-hybrid assay are covered [(1) bait-AMPK subunit and
prey-interactor; (2) bait-interactor and prey-AMPK subunit],
since sometimes one direction gives stronger results that the
other. The GAD-based constructs become completely neces-
sary when a particular AMPK subunit shows signs of self-
activating properties when fused to LexA (seeNote 9). - Since the yeast strain contains four usable different auxotro-
phies (trp1, leu2, ura3andhis3), up to two additional plasmids
carrying the selection markersURA3andHIS3can be intro-
duced in the cells to regulate the interaction mediated by the
bait (TRP1) and prey (LEU2) plasmids. In Fig.4, it is shown
that the expression of a third protein (protein F) improves the
interaction between AMPKα2 and AMPKβ1. - Alternative yeast strain can be used in the yeast two-hybrid
assay. For example CTY10.5d (MATa ade2 his3 leu2 trp1
ga 4 gal80 URA3::lexAop-lacZ)or TAT7 (MATa ade2 his3
leu2 trp1 ga4 gal80 LYS2::lexAop-HIS3 URA3::lexAop-lacZ)
[11, 28]. However, in our hands the THY-AP4 strain gives
better performance that the other ones. - In the yeast two-hybrid assay it is essential to analyze if the bait
construct by itself has self-activating properties. In other
words, if the bait plasmid by itself is able to activate the tran-
scription of the reporter genes. In the case of AMPK, bait
plasmids such as LexA-AMPKα1, LexA-AMPKα2, LexA-
AMPKβ2, LexA-AMPKγ1 and LexA-AMPKγ2 give negative
results on self-activation (Fig.3a). However, LexA-AMPKβ 1
has a weak self-activating performance (Fig.3a) and LexA-
154 Pascual Sanz et al.