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Figure 1.4 Subcellular movement of PIN proteins. Schematic model to explain internalization of PIN proteins upon BFA treatment. BFA blocks GNOM ARF-GEF responsible for activation of endosomal ARF GTPases, which mediate recycling of PIN1 to the plasma membrane. Ongoing endocytosis is BFA insensitive. AEIs such as TIBA interfere with both steps of PIN cycling.

BFA are guanine nucleotide exchange factors (GEFs), which activate small GTPases of the ARF family - important regulators of vesicle budding. In Arabidopsis roots, PIN1 protein is rapidly and reversibly internalized from the plasma membrane in response to BFA (Fig. 1.4) (Geldner et al, 2001). This also occurs in the presence of a protein synthesis inhibitor, thereby demonstrating that internalized PIN1 originated from the plasma membrane and that PIN1 is rapidly cycling between the plasma membrane and a 'BFA' compartment, recently characterized as an accumulation of endosomes (Plate 1.1E) (Geldner et al., 2003). The action of drugs that disrupt the structure of the cytoskeleton indicated that PIN1-containing vesicles are transported predominantly along the actin cytoskeleton. However, in dividing cells, tubulin is also required for correct PIN1 traffic (Geldner et al., 2001). The actin-dependent recycling was also demonstrated for PIN3 (Friml et al., 2002a) and PIN2 (Grebe et al., 2003) proteins. The PIN recycling phenomenon has been further corroborated by electron microscopy studies, which detected PIN3 not only at the plasma membrane but frequently also in intracellular vesicles (Friml et al., 2002a). BFA is also known to rapidly interfere with auxin efflux and could phenocopy the effect of AEIs (Delbarre etal., 1998; Morris & Robinson, 1998; Geldner et al, 2001). These surprising findings can be hardly incorporated in the old static models, with influx and efflux carrier complexes residing and functioning at the plasma membrane. The crucial question remains of what the biological relevance of the PIN cycling is. Here different scenarios can be conceived:

1. A high turnover of PAT components would provide the flexibility to allow rapid changes in polarity of carrier distribution and provide a mechanism for the rapid redirection of auxin fluxes in response to environmental or developmental cues (Friml & Palme, 2002). Indeed, PIN3 was shown to rapidly relocate in response to gravity stimulation (Plate 1.1M) (Friml et al, 2002a).

2. Components of polar auxin transport may have a dual receptor/transporter function (Hertel, 1983). In this case, cycling may be part of a mechanism for signal transduction and receptor regeneration, as is known for some other kinds of receptors. Dual sensor and transport functions have been proposed for sugar transporters in yeast and plants (Lalonde et al., 1999).

3. The most exciting possibility is that vesicle trafficking itself is a part of the PAT mechanism and that, in a manner analogous to the mechanism of neurotransmitter release in animals, auxin is a vesicle cargo, released from cells by polar exocytosis (Friml & Palme, 2002). In this model, instead of being 'auxin channels', PIN proteins would mediate the accumulation or retention of auxin in the vesicles in which auxin would be translocated. Some support of this scenario comes from the BIG protein, which is involved in PAT and PIN1 subcellular trafficking, since its homolog in Drosophila mediates vesicle recycling during synaptic transmission.

Regardless of how well any of the scenarios described above eventually turns out to fit the true picture, an understanding of the cellular mechanisms controlling the subcellular dynamics of the auxin carriers will be crucial for our understanding of PAT process.

1.4.2 AEIs and vesicle trafficking

Another surprising outcome of the cell-biological studies on PIN cycling concerns effects of AEIs such as TIBA on vesicle trafficking (Fig. 1.4). Despite the fact that AEIs were major tools for physiological studies on PAT, the mechanism of their action remains elusive. The finding that TIBA inhibits PIN1 recycling (Geldner et al., 2001) raised an attractive possibility that TIBA inhibits auxin efflux by interfering with the recycling of auxin efflux components. However, AEIs also interfere with vesicle-mediated traffic of PAT unrelated proteins and much higher concentrations are needed for trafficking inhibition than for PAT inhibition. In addition, observations on BY-2 tobacco cells have revealed that the inhibition of auxin efflux by NPA is much more efficient than the inhibition caused by the well-established inhibitor of protein traffic BFA (Petrasek et al., 2003). These findings argue against a causal link between a general role of NPA and other phytotropins in vesicle trafficking and auxin efflux inhibition. Thus, it still remains open whether inhibition of vesicle traffic and PAT by AEIs are functionally related.

1.4.3 GNOM and PIN dynamics

Significant complementary evidence for the importance of subcellular vesicle trafficking in the process of PAT, and especially for its importance in plant development, came from the analysis of the Arabidopsis mutant gnom. The gnom (gn) mutant (Plate 1.1G) was isolated from a screen for defects in early apical-basal patterning in Arabidopsis seedlings (Mayer et al., 1991). Embryos of gn mutants display a variety of aberrations, including missing roots and fused or improperly placed cotyledons. Most of these defects are reminiscent to defects observed when embryos are cultivated in the presence of polar auxin transport inhibitors (Hadfi et al., 1998; Friml et al., 2003). The GN gene was identified as a GEF for ARF (ADP-ribosylation factor) GTPases - essential regulators of vesicle trafficking in many organisms. ARFs participate in the formation of transport vesicles from donor compartments and the selection of their protein cargo. ARF proteins are present in two forms: an active GTP-bound and an inactive GDP-bound form. Conversion of GDP-bound to the GTP-bound form is mediated by specific GEFs (Donaldson & Jackson, 2000). The function of ARF-GEFs such as GNOM is inhibited by BFA, which binds to an ARF-GDP/ARF-GEF complex (Peyroche et al., 1999). At first, it was difficult to reconcile the biochemical function of GNOM in vesicle trafficking with the auxin-transport-related phenotype of the gnom mutant. A detailed analysis of gn revealed that at the subcellular level, the coordinated polar localization of PIN1 was defective in gnom mutant embryos (Steinmann et al., 1999). This finding, taken together with a BFA-sensitive subcellular cycling of PIN proteins, suggested a role for GNOM ARF-GEF in the regulation of subcellular trafficking of PAT components such as PIN proteins (Fig. 1.4). Consistently with this, GNOM localizes to and maintains the integrity of endosomes through which PIN proteins recycle (Geldner et al., 2003). To determine whether GNOM specifically controls PIN1 traffic, a single amino acid substitution (696M to L) was introduced into the originally BFA-sensitive GNOM, to generate a fully functional, but BFA-insensitive variant of the GNOM protein. This allowed specifically dissecting the function of GNOM from other BFA-sensitive trafficking steps. When plants expressing the BFA-resistant GNOM are treated with BFA, PIN1 remains correctly localized to the cell surface (Fig. 1.4), demonstrating direct involvement of GNOM in PIN1 recycling. In addition, BFA-resistant GNOM renders both auxin efflux and auxin-mediated growth insensitive to BFA inhibition (Geldner et al., 2003). Thus, these findings directly linked a component of membrane traffic - GNOM ARF-GEF to the PIN recycling and PAT process, highlighting the importance of polarized trafficking in fundamental processes of plant development.

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