Traffic

Directed vesicle traffic is essential for maintaining cell polarity, growth and development in plants, and is responsible for delivery of some of the most biologically interesting and commercially important products, including various alkaloids, anticancer drugs, dyes and enzymes [cf. Walker (2003) Plant Physiol. 132,44]. The superfamily of membrane and membrane-associated proteins known as SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) are key components of the mechanics for membrane vesicle traffic. Subsets of SNAREs occur at vesicle and target membranes and interact to form a tetrameric bundle of coiled helices which draws the membrane surfaces together and thus provides a mechanism for recognition, docking and fusion [see Bassham and Blatt (2008) Plant Physiol. 147,1504].

We are currently exploring SNARE function, taking advantage of advanced tagging technologies, including use of photo-activatable and photochromic markers, including paGFP and EOS, to monitor the traffic of specific soluble and intrinsic membrane proteins. To test SNARE function, we use a combination of dominant-negative and rescue strategies in vivo [see Geelen et al. (2002) Plant Cell 14, 387-406; Sutter et al. (2006) Plant Cell 18,935-54; Honsbein et al. (2009) Plant Cell 21,2859] that faithfully recapitulate the known interactions (and lack thereof) between SNAREs [Tyrrell, et al. (2007) Plant J. 51, 1099-1115].

With these needs in mind, we developed a toolchest of Gateway-compatible plasmid vectors and molecular markers for use in Arabidopsis, taking advantage of the Ubiquitin-10 promoter to reduce endogenous suppression mechanisms in the plant, and we advanced methodologies for transient transformation of Arabidopsis seedlings with Agrobacterium tumefaciens GV3101 [Grefen, et al. (2010) Plant J. 64,355]. Additionally, we recently introduced a set of bicistronic vectors for coexpresson of the inserted coding sequences from a single vector backbone [Chen, et al. (2011) Plant Cell Envir., 34:554]. Check here for further details about the vector set and their applications.

Our work demonstrated that the SNARE SYP121 (=NtSYR1) of tobacco and its Arabidopsis homologue SYP121 (=AtSYR1/PEN1) function late in trafficking from the trans-Golgi network to the plasma membrane [Geelen et al. (2002); Sutter et al. (2006); Tyrrell et al. (2007)]. Intriguingly, some cargos reach the plasma membrane via one or more pathways independent of the SNARE [Sutter et al. (2006)]. Thus, traffic to the plasma membrane diverges at some point post-Golgi.

These studies also uncovered a role for the plasma membrane SNAREs in anchoring the KAT1 K⁺ channel. The anchoring of proteins in complexes is one of the most important features common to metabolic and signalling networks, and is thought to facilitate signal transmission within the complex. Such complexes are often localised within so-called lipid rafts. We found [Sutter, et al. (2006)] that KAT1 assembles within microdomains, evident as dense, 0.5-µm clusters and associated with lipid rafts. KAT1 showed a lateral diffusion constant at least two orders of magnitude less than cytoskeletally-anchored proteins such as the Band-3 protein of erythrocytes. Both export of KAT1 and its microdomain anchoring depended on SYP121. This loss of microdomain organization might be explained simply as a consequence of changes in KAT1 delivery to the plasma membrane. However, the KAT1 protein remaining within the plasma membrane showed roughly a 100-fold increase in lateral mobility. Thus, it seems that the SNARE is important for anchoring KAT1 within the microdomains.

Finally, we have found KAT1 microdomains also to serve as focal points for K⁺ channel endocytosis and its recycling back to the plasma membrane. Not only does ABA affect channel activity, but it also triggers a selective endocytosis and sequestration of the K⁺ channel within an endosomal membrane pool [Sutter et al. (2007) Curr. Biol. 17,1396-1402]. These studies set two important precedents for plants, first in demonstrating an evoked and selective endocytosis of the protein, and second in providing unambiguous evidence for its subsequent recycling back to the plasma membrane. As the KAT1 K⁺ channel normally is active in K⁺ uptake for stomatal opening, the data imply a role for channel traffic in accommodating long-term changes in osmotic solute transport that enable guard cells to adapt to repeated environmental challenges. Thus, we can speculate that such submicroscopic organisation is a feature essential for K⁺ channel regulation mediated both by channel gating and by channel protein traffic.