The Muday Laboratory, Wake Forest University

Localization of Auxin Transport Proteins

The polarity of auxin transport is driven by the asymmetric localization of the auxinlocalization of auxin transport protiens (micrograph) efflux carrier complexes to one side of auxin transporting cells, which thereby control the unidirectional movement of auxin. The localization of a green fluorescent protein fusion to an IAA transport protein (PIN1) shows this polarity in the roots of Arabidopsis in the associated image. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through endocytotic cycling of membrane proteins, while the attachment of an efflux carrier subunit to the actin cytoskeleton may facilitate this cycling (as reviewed in Muday et al. 2003).

We have been examining the molecular mechanisms that control the localization of auxin transport proteins using several mutants with defects in proteins predicted to regulate this process. In collaboration with Dr. Leslie Sieberth at the University of Utah, we have looked at the scarface mutant which has a defect in a protein that regulates the activity of small G proteins that may modulate vesicle movement. We have found that this mutant has altered auxin transport consistent with altered localization of PIN1-GFP and leaf and root development (Sieberth et al., 2006). We are also examining the role of the temperature sensitive allele of scd1 in regulation of auxin transport and root development and gravitropism in collaboration with Dr. Tanya Falbel at the University of Wisconsin. SCD1 encodes a protein whose sequence predicts an interaction with RAB proteins, a class of small G-proteins. These experiments are focused on examination of auxin transport and PIN1-GFP localization under conditions where SCD1 protein does not function.We have been exploring one protein that is part of the auxin efflux carrier complex, the NPA binding protein, which is the site of action of auxin transport inhibitors, including NPA (naphthylphthalamic acid). We have found that the NPA binding protein partitions with the actin cytoskeleton during detergent extraction and in vitro depolymerization and repolymerization of actin and that treatments that fragment the actin cytoskeleton release NPA binding activity from the cytoskeleton and reduce auxin transport (Butler et al., 1998). Finally, the NPA binding protein is retained by F-actin affinity columns, indicating a direct interaction between the NPA binding protein and the actin cytoskeleton (Hu et al., 2000). Currently, efforts are focused on characterizing the interaction between the NPA binding protein and other proteins that make up the auxin efflux carrier complex.

(graduate student names in bold, undergraduates underlined)

Butler, JA, S Hu, S Brady, MW Dixon and GK Muday (1998) In vitro and in vivo evidence for actin association of the NPA binding protein. Plant Journal 13, 291-301.

Hu S, Brady, SR, Kovar, D, Staiger, C, Clark, G, Roux, S, and Muday, GK. (2000) Identification of plant actin binding proteins by F- actin affinity chromatography. Plant Journal 24: 127-137

Muday, GK, Peer, WA, Murphy, AS (2003) Vesicular cycling mechanisms that control auxin transport polarity. Trends in Plant Science. 8:301-303.

Sieberth, LE, Muday, GK, King, EJ, Benton, G, Kim, S, Metcalf, KE, Myers, L, and Seamen, E (2006) Scarface encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis. Plant Cell 18: 1396-1411

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