Date of Award
Doctor of Philosophy
Theses & dissertations (Interdisciplinary)
Diane C. Bassham
Steve A. Whitham
David J. Oliver
Sorting nexins are conserved proteins that function in vesicular trafficking and are defined by the presence of a phox (PX) domain. Here we present the characterization of Arabidopsis thaliana sorting nexin AtSNX2b. The ubiquitously-expressed sorting nexin is a peripherally-associated membrane protein whose PX domain binds to phosphatidylinositol 3-phosphate in vitro. AtSNX2b localizes to the trans-Golgi network (TGN), prevacuolar compartment and endosomes. Overexpression of GFP-tagged AtSNX2b produces enlarged GFP-labeled compartments that can be also labeled by the endocytic tracer FM4-64, while untagged AtSNX2b overexpression generates compartments which are not as large. Endocytic trafficking of FM4-64 is arrested in cells overexpressing AtSNX2b or GFP-AtSNX2b, while overexpression of AtSNX2b interferes with the transport to the vacuole of proteins containing an N-terminal vacuolar sorting signal but does not affect proteins containing C-terminal vacuolar sorting signal. The intracellular function of AtSNX2b is hypothesized to be in vesicular trafficking from endosomes to the vacuole.;Over-expression and null mutant plants of AtSNX2b show enhanced or reduced root growth, respectively, under nutrient stress conditions during early development of seedlings. Atsnx2b knockout (KO) mutant root growth is slowed on nitrogen-deficient, carbon-deficient and high sucrose stress media. Because the Atsnx2b KO can be affected by both carbon-deficiency and carbon abundance, it is possible that the AtSNX2b protein contributes to or regulates root growth in developing plants.;To further understand the function of AtSNX2b in vesicular or endosomal trafficking, potential AtSNX2b-interacting factors were examined. Sorting nexins can form homo- and hetero-dimers with other sorting nexins. AtSNX2b colocalizes with AtSNX1 on endosomal structures, suggesting a potential AtSNX1-AtSNX2b dimer. The analysis of other endosomal localized proteins demonstrated localization of AtSNX2b to BRI1-labeled endosomal structures and this association was supported by detection of AtSNX2b in BRI1-GFP immunoprecipitations. Evidence for TGN function of AtSNX2b was provided by the detection of AtSNX2b in immunoprecipitates of the TGN t-SNARE T7-SYP42. Finally, the analysis of AtSNX2b-interacting factors by co-immunoprecipitation revealed three potential associated proteins that are not predicted to be trafficking components. These proteins are potential cargoes for the AtSNX2b complex and further investigation will determine their function.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Phan, Nguyen, "Functional characterization of Arabidopsis sorting nexin AtSNX2b" (2008). Retrospective Theses and Dissertations. 15631.