Transcript Slide 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unpublished research showed that the Arabidopsis sorting nexin AtSNX2b is present in several intracellular organelles including the trans-Golgi network, prevacuolar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.
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
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.
Plasmid Construction.
cDNA
pGEM
Restriction digest
SalI Site
Insert/ Gene of Interest
BamHI Site
Figure 2. Expression of pDGmR::SNX2a in
Suspension Cells. This figure shows Arabidopsis
Figure 3. Expression of Control in Suspension
Cells. This figure shows a Arabidopsis suspension cell
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.
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.
170.8 –
1 2 3 4
109.5 -78.9 --
Vector with Fluorescent protein
60.4 --
Plant 1
A
B C
Plant 3
A
B C
47.2 –
SalI Site
BamHI Site
References.
1. Carlton, J., Bujny, M., Rutherford, A. and Cullen, P. (2004). Sorting Nexins –
Unifying Trends and New perspectives. Traffic 2005 6: 75-82.
35.1 –
2. Sato, T.K., Overduin, M., Emr, S.D. (2001). Location, Location, Location: Membrane
Targeting Directed by PX Domains. Science 294: 1881-1885.
24.9 –
Ligation
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.
18.3 –
3. Sheen, J. (2002) A transient expression assay using Arabidopsis mesophyll
protoplasts. <http://genetics.mgh.harvard.edu/sheenweb/>
13.7 –
(kDa)
Figure 4. Western Blot Analysis of
the Transformed Protoplasts. This
Insert
SalI Site
BamHI site
Vector
Transformation into DH5α
cells
Antibiotic Resistant Colonies
Containing plasmid with correct
insert
Transformation into
Arabidopsis Protoplasts
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.
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 TDNA 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.
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.
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.
Acknowledgements.
This research was supported by National Science Foundation – Research Experience for
Undergraduates and Iowa State University.