Auxin as an intercellular signal - Intercellular Communication in Plants - page 6

suggested the existence of an NPA-binding protein (NPB), forming a part of the
auxin efflux carrier complex (Fig. 1.3). Despite extensive studies on the NPB, little
is known about its nature. Studies on NPB revealed different localizations for NPB,
either at the periphery of the plasma membrane associated with the cytoskeleton
(Cox & Muday, 1994) or as an integral membrane protein (Bernasconi et al ., 1996).
On the other hand, several proteins, which bind NPA with different affinities, have
been isolated from Arabidopsis protein extracts by NPA affinity chromatography.
These include multidrug resistance (MDR) proteins of the ATP-binding cassette
(ABC) transporter family (AtMDR1 and AtPGP1), aminopeptidase (AMP1) and
others (Murphy et al ., 2002). In addition, an Arabidopsis mutant named tir3 was
isolated in a genetic screen for NPA-insensitive roots. This mutant was shown to
have reduced NPA-binding activity and auxin transport capacity, suggesting the
possibility that TIR3 encodes the NPB or a functionally related protein (Ruegger
et al ., 1997). Nonetheless, the characterization of the TIR3 gene product as a ho-
molog of Drosophila CALOSSIN/PUSHOVER (CAL/O) protein, termed BIG for
its extraordinary size (Gil et al ., 2001), did not clarify its functional connection to
auxin efflux or NPA binding. In summary, both biochemical and genetic approaches
provided candidates for the NPB, but the identity of the NPB and the mechanism by
which NPA inhibits polar auxin transport still remains a matter of debate. Based on
additional experiments using protein synthesis inhibitors, such as cycloheximide,
the existence of a third unstable component of the auxin efflux carrier complex has
been proposed (Fig. 1.3). Short-term cycloheximide treatment does not affect auxin
efflux or the saturable binding of NPA to microsomal membranes, but it interferes
with the inhibitory effect of NPA on efflux (Morris et al ., 1991). These observa-
tions suggest that the NBP and the efflux carrier itself are separate proteins with
low turnover, interacting through a third, unstable protein (Delbarre et al ., 1998;
discussed by Morris, 2000). Surprisingly, inhibitors of vesicle traffic to the plasma
membrane, monensin and Brefeldin A (BFA), interfere with cellular auxin efflux
(Delbarre et al ., 1998; Morris, 2000) and with polar (basipetal) auxin transport in
stem segments (Robinson et al. , 1999). Significantly, BFA does not affect saturable
NPA binding to microsomal preparations, providing additional evidence that the
NBP and the auxin efflux catalyst are different proteins (Robinson et al. , 1999).
Taken together, these observations indirectly suggest that an essential component of
the auxin efflux carrier system, but not the NPB, is targeted to the plasma membrane
through the BFA-sensitive secretory system, and that this component turns over very
rapidly at the plasma membrane without the need for concurrent protein synthesis.
This implies that part of auxin efflux carrier component rapidly cycles between the
plasma membrane and internal pools (Delbarre et al ., 1998; Morris & Robinson,
1998; Robinson et al. , 1999).

1.3 Molecular components

Most instrumental in identifying molecular components of PAT were genetic ap-
proaches, especially in Arabidopsis thaliana .Various screening strategies have been