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as bait. Such a sequential multi-round Y2H screening approach is termed
''interactome walking'', which can identify novel interactors linking distinct func-
tions (
Cusick et al., 2005
). In this study, they identified 71 interactions among
59 proteins, comprising a complex interactome map. Because Y2H screens might
not reflect the in vivo functional relationships between two genes, other functional
genomic techniques and conventional genetic approaches are often used for indepen-
dent experimental validation. In this study, the identified interactions were confirmed
by coaffinity purification assays and functionally validated by double genetic pertur-
bation analysis in which RNAi was used to inactivate genes in loss-of-function mutant
backgrounds of known daf-7/TGF-
b
pathway genes. Through these approaches, nine
genes were confirmed to interact with the daf-7/TGF-
b
pathway, eight of which had
not previously been reported to have roles in the daf-7/TGF-
b
pathway. Given the high
false-negative rate (approximately 60-70%) of Y2H screens (
Wa lhou t et al., 2000
)
and intrinsic limitations of RNAi (discussed above), they speculated that many more
interactors were likely missed in this study. Nevertheless, this study highlights the
power of coupling large-scale protein interaction mapping with conventional genetic
perturbation to identify components of signaling pathways in C. elegans.
Another strategy for identifying interacting proteins is affinity-based protein
isolation coupled with MS analysis (
Aebersold and Mann, 2003
). This strategy is
used to identify proteins that are associated with a protein of interest. These asso-
ciated proteins can be isolated through two affinity purification techniques: (1)
immunoprecipitation, an antibody-based purification, in which an antibody against
a protein of interest is used to isolate it and its associated proteins; (2) tandem
affinity purification (TAP), in which two tags separated by an enzyme-cleavable
site are fused to a protein of interest and the associated proteins are isolated in two
steps sequentially using antibodies or binding proteins against the two tags (
Fig. 6
).
The tags can be fluorescent proteins, such as GFP, which allow both dynamic
imaging studies and proteomic analysis. This type of tag is also referred to as the
''localization and affinity purification'' (LAP) tag (
Cheeseman and Desai, 2005;
Rigaut et al., 1999
). Compared with the Y2H technique in which the proteins cannot
undergo some of the post-translation modifications required for particular interac-
tions in metazoans, the tag-based strategy has several advantages: (1) the fully
processed and modified protein can be used as a bait; (2) bound proteins are isolated
from the native cellular environment where interactions take place; (3) multiple
associated components can be isolated and analyzed at a single time (
Ashman et al.,
2001
). An example of the application of TAP/LAP coupled with MS for identifying
genes was a proteomic study on C. elegans kinetochores (
Cheeseman et al., 2004
), a
specialized organelle that regulates chromosome segregation in mitosis and meiosis
(
Maiato et al., 2004
). To isolate the proteins involved in the assembly and function of
kinetochores, they first generated a transgenetic strain expressing two newly iden-
tified kinetochore proteins that were used as bait and fused with two tags, GFP and
the S peptide domain. These two tags were separated by a sequence recognized by
the tobacco etch virus (TEV) protease. Two sequential rounds of affinity isolation
were performed. The bound proteins were first isolated using an antibody against
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