3.3 Approaches to identify proteins that mediate IAA efflux
Biochemical and genetic approaches have identified a number of proteins associated with
auxin transport. Auxin transport inhibitors have been used in both of these approaches.
Auxin efflux inhibitors reduce polar IAA transport, efflux of auxin from membranes and
cells, and inhibit physiological processes dependent upon auxin transport, such as gravi-
tropic curvature (as reviewed in Rubery 1990). In particular, the inhibitor, naphthylph-
thalamic acid (NPA), has been used in biochemical experiments, since a tritiated form of
this molecule can be used to follow the activity of one class of auxin transport proteins,
termed NPA binding proteins (Muday et al. 1993). These proteins have been biochemi-
cally characterized using their NPA binding activity (Dixon et al. 1996; Butler et al. 1998;
Hu et al. 2000, as reviewed in Muday 2000).
Recently, NPA affinity chromatography has been used to identify a number of proteins
that bind NPA (Murphy et al. 2002) with some proteins identified by both this method
and genetic approaches (Noh et al. 2001), as described below. More recently, auxin in-
flux inhibitors have also been identified (Imhoff et al. 2000; Rahman et al. 2001a; Parry
et al. 2001a), but as yet these inhibitors have been used to characterize previously identi-
fied proteins, not to identify new proteins.
Genetic approaches have been the most productive in identifying candidates for auxin
transporters and associated regulatory proteins. The screens that have identified these
proteins have included altered responses to auxin (Maher and Martindale 1980) or to
auxin transport inhibitors (Ruegger et al. 1997), altered growth and developmental
processes that are dependent upon auxin transport, including gravity response (Chen et
al. 1998), and a number of developmental processes (Okada et al. 1991; Noh et al. 2001).
Many of the mutant genes have been identified and the functions of the encoded proteins
have been linked to IAA influx or efflux, as described below.
3.4 Proteins that mediate IAA efflux
Experimental evidence suggests that IAA efflux carriers are made of multiple protein
subunits, which are encoded by gene families with unique expression patterns that medi-
ate the distinct polarities of IAA movement in different tissues (as reviewed by Blakeslee
et al. 2005). The first gene family linked to IAA efflux encodes PIN proteins (as reviewed
by Friml 2003). Arabidopsismutants with defects in PIN1 were isolated due to their pin-
formed inflorescences and were shown to have reduced IAA transport in this tissue
(Okada et al. 1991). PIN1 encodes a protein with 10 membrane-spanning domains, which
is asymmetrically localized to the basal end of the membrane of inflorescence cells, con-
sistent with a role in mediating basipetal IAA transport (Gälweiler et al. 1998). Several
other screens isolated pin2/agr1/eir1/wav6mutants based on altered root growth in re-
sponse to gravity, waving, or ethylene and found that these mutant genes encoded a pro-
tein with extensive sequence similarity to PIN1 (Chen et al. 1998; Müller et al. 1998;
Luschnig et al. 1998; Utsuno et al. 1998). These mutants have reduced basipetal IAA
transport in roots (Chen et al. 1998; Rashotte et al. 2000) and the PIN2 protein is ex-