Investigation of Arabidopsis thaliana proteins AtSNX2a and AtSNX1

1. Investigation of Arabidopsis thaliana proteins AtSNX2a and AtSNX1 Lucas Greder and Diane Bassham, National Science Foundation-Research Experience for Undergraduates, Iowa State University, Ames, Iowa Abstract.Phylogenetic analysis of several plant and animal sorting nexins (Vanoosthuyse, et al. 2003) showed three related Arabidopsis sorting nexins, AtSNX2a, AtSNX2b, and AtSNX1, as being similar to Brassica sorting nexin 1. Previous unpublished research showed that the Arabidopsis sorting nexin AtSNX2b is present in several intracellular organelles including the trans-Golgi network, prevacuolar compartment and in endosomes and likely functions in membrane trafficking and protein sorting. We hypothesize that AtSNX2a and AtSNX1 may have a similar role in vesicle trafficking in Arabidopsis. The objective of this project involved finding the location and determining the function of AtSNX2a and AtSNX1. In order to determine the location of the proteins in the cells, fusion proteins with fluorescent proteins were assembled for each gene and transformed into Arabidopsis thaliana protoplasts. Cells transformed with the pGDmR::AtSNX2a construct showed expression but did not provide evidence for the location of the native protein. Cells transformed with pGDmR::AtSNX1 construct did not show any expression. In order to begin functional analysis of the genes, plants were screened by PCR for knockout insertions consisting of T-DNA inserts inside the gene of interest. Plants that were homozygous for the insertion were identified and will be used for phenotypic analysis. Introduction.Efficient trafficking of macromolecules within cells is necessary for proper cell function. Sorting nexin proteins help the cell move proteins to the correct location within the endomembrane system. A region of the sorting nexin protein called a PX domain allows it to be directly targeted to specific locations that contain a phosphatidylinositol phospholipid. After the PX domain binds to the PIP on the membrane, other reactions can take place, including binding to other proteins, regulating protein complexes, cytoskeletal organization, protein phosphorylation, lipid modification and other biological process (Sato et al., 2001). The case for evolutionary conservation from species to species relies on the retention of the PX domain, a 100-300 amino acid sequence. The mammalian sorting nexin, SNX2, is a homologue of the yeast protein VPS5 which has been shown to aid in protein trafficking (Worby, et al. 2002). The sorting nexin SNX1, is thought to perform a similar role to VPS5 as a regulator of hydrolase receptor transport from the endosomal system (Carlton, et al. 2004). The presence of the PX domain and the function described from previously unpublished research suggests that the Arabidopsis protein AtSNX2b may play a role in vesicle trafficking and therefore AtSNX2a may play a similar role. The similarity of the AtSNX1 amino acid sequence to a Brassica sorting nexin suggests that AtSNX1 may also function as a sorting nexin in Arabidopsis. Data. Summary.Possible interpretations of localization results: 1. Only the RFP (pGDmR) is being made, either due to a problem with the construct, or because the full length protein is made but is being partially degraded. 2. The fusion protein is made correctly but is mislocalized due to the fusion with RFP. 3. The sorting nexin is actually localized to the cytosol, even though we expected it to be associated with membranes. Comparing figure 2 to figure 3, both the control and the fusion protein appear to be cytosolic. No definitive conclusion can therefore be made about the location of the expressed fusion protein. The gene of interest was cloned into the plasmid behind the fluorescent protein (A). This orientation may affect the N-terminus of the protein in a way that affects the protein function. Future research should involve cloning the gene of interest into the construct in front of the fluorescent protein (B) in order to limit the possible effect that the fluorescent protein may have on location and degradation of the protein. Transformed Arabidopsis Leaf Cell Protoplast Figure 1. Expression of pGDmR::SNX2a in Leaf Cells.This figure shows an Arabidopsis leaf cell transformed with pGDmR::SNX2a. Picture A is the cell in the bright field lighting, picture B is the cell under green fluorescence and picture C is the cell under red fluorescence. The pGDmR vector contains a red fluorescence tag, dsRed, which allows the expression to be seen in picture C. Picture B shows the natural fluorescence coming from the chloroplasts. The difference between picture B and picture C represents the location of the expressed pGDmR::SNX2a. The expression is localized to the cytoplasm without a specific membrane location. Transformed Arabidopsis Suspension Cell Protoplasts Plasmid Construction. cDNA pGEM Figure 2. Expression of pDGmR::SNX2a in Suspension Cells. This figure shows Arabidopsis suspension cell protoplasts transformed with the pGDmR::SNX2a plasmid. Picture A shows the transformed cell’s red fluorescence and picture B shows the transformed cell along with other untransformed suspension cells. Figure 3.Expression of Control in Suspension Cells. This figure shows a Arabidopsis suspension cell protoplasts transformed with the pGDmR vector control alone. The similarities between figure 3A and figure 2A suggest that the AtSNX2a protein is not being correctly expressed or localized. In figure 5, plant 3 is homozygous for the knockout. Based on previous knowledge and research, it would prove insightful to determine the effect the knockout mutation might have on trafficking of marker proteins. Restriction digest 1 2 3 4 SalI Site Insert/ Gene of Interest BamHI Site 170.8 – 109.5 -- 78.9 -- 60.4 -- 47.2 – 35.1 – 24.9 – 18.3 – 13.7 – (kDa) Plant 1 Plant 3 A B C A B C References. 1. Carlton, J., Bujny, M., Rutherford, A. and Cullen, P. (2004). Sorting Nexins – Unifying Trends and New perspectives. Traffic 2005 6: 75-82. 2. Sato, T.K., Overduin, M., Emr, S.D. (2001). Location, Location, Location: Membrane Targeting Directed by PX Domains. Science 294: 1881-1885. 3. Sheen, J. (2002) A transient expression assay using Arabidopsis mesophyll protoplasts. <http://genetics.mgh.harvard.edu/sheenweb/> 4. Vanoosthuyse, V., Tichtinsky, G., Dumas, C., Gaude, T., Cock, J. M. (2003) Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphotase with Brassica oleracea S Locus Receptor Kinase. Plant Physiology 133: 919-929. 5. Worby, C.A., Dixon, J. E. (2002). Sorting Out The Cellular Functions of Sorting Nexins. Nature Reviews 3: 919-931. Vector with Fluorescent protein SalI Site BamHI Site Ligation Figure 5. PCR Results from T-DNA Screening.This figure shows the 33351 T-DNA insert present in the AtSNX1 Salk line. Each plant DNA was run with 3 combinations of primers, left and right full length primers (A), the left full length primer with the T-DNA insert primer (B), and the right full length primer with the T-DNA insert primer (C). Plant 1: The results suggest that this plant is heterozygous for the T-DNA insertion. There is a band for the full length (Column A) and there are bands for the LP-LBb1 and RP-LBb1 columns (B & C). Plant 3: This plant is homozygous for the T-DNA insertion. There is no band present for the full length primers which indicates that the T-DNA is inserted within the gene and there are bands for the LP-LBb1 and RP-LBb1 primers. Figure 4. Western Blot Analysis of the Transformed Protoplasts. This figure shows the western blot analysis of the transformed protoplasts. For the fusion protein (lanes 1 and 2), pGDmR::SNX2a, a band of around 90 kDa is expected. However, the only lanes that contained specific bands are the lanes with the controls (lanes 3 and 4). These bands are around 30 kDa in size. Insert Transformation into DH5α cells BamHI site SalI Site Antibiotic Resistant Colonies Containing plasmid with correct insert Vector Acknowledgements. This research was supported by National Science Foundation – Research Experience for Undergraduates and Iowa State University. Diagram 1. T-DNA Insert (right). In order to determine the function of the proteins of interest the plants must be screened for knockouts. The T-DNA inserts are inserted into the gene of interest and are designed to inhibit the gene from functioning properly. A plant that is homozygous for the T-DNA insertion will not express the gene of interest. Transformation into Arabidopsis Protoplasts