1. The authors aimed to determine if the Arabidopsis thaliana p80 protein localizes to endosomal vesicles like its animal homolog by expressing a GFP fusion of the plant protein in HeLa cells. 2. Control fusion proteins expressed well but the plant p80 protein did not reach sufficient levels for localization analysis, only showing background fluorescence. 3. Low transfection efficiency of the plant protein into human cells was a challenge, suggesting a plant-based system would be better for future studies of p80 protein localization.

1. Expression and localization of Arabidopsis thaliana p80 protein in a human cancer cell line
Joseph S. Danner and F. Les Erickson, Ph.D.
Department of Biological Sciences, Salisbury University, Salisbury, Maryland
Abstract
We have identified a gene in the model organism Arabidopsis thaliana whose gene product is believed to play a role in endosomal
recycling. This gene, termed p80, is thought to have an ortholog present in animal cells where it has been implicated in
downregulation of T-cell receptor signaling (3), a process linked to the trafficking of endosomal vesicles. Strong sequence
similarities, protein interactions, and structural motifs provide a strong basis for the idea that these homologous genes encode
proteins of comparable function (Erickson, unpublished results). To substantiate the hypothesis that p80 functions in endosomal
trafficking, fluorescence microscopy is used to identify the localization of the p80 protein in immortalized HeLa cells. An
expression plasmid is used to introduce p80 into HeLa cells through transient transfection methods. Expression of the plasmid then
yields a fusion protein in which p80 is conjugated to GFP; localization can then be detected by using techniques in fluorescence.
Conclusions
•Control gene constructs -GABARAP, GFP, Actin, and Myosin III- all show robust expression in HeLa cells.
•Plant p80 does not express to levels sufficient for determining if it localizes to endosomal vesicles.
•Low expression levels resulted in only background levels of fluorescence in p80 transformed cells. This was misleading at first because it looked as
if p80 protein localized to vesicles (see fig 2.P-R ). Untransformed control cells (see fig 2.L-N), however, show the same fluorescent patterns,
indicating that p80 is not being expressed to sufficient levels in this heterologous system.
•Low transfection levels raise concern that the protocol used may be inefficient. Between 30 and 50 percent of all cells subjected to transfection
showed expression; these levels varied in intensity.
•A plant based transfection system should prove more valuable in future studies.
References
1. Erickson, F.L., Corsa, A.C., Dosé, A.C., Burnside, B. 2003. Localization of a Class III Myosin to Filopodia Tips in Transfected HeLa Cells Requires
an Actin-binding Site in its Tail Domain. Molecular Biology of the Cell 14:4173-4180.
2. Green, F., O’Hare, T., Blackwell, A., Enns, C.A. 2002. Association of human transferrin receptor with GABARAP. FEBS Letters 518(1-3):101-06
3. Park, J., B.S. Lee, J.K Choi, R.E. Means, J. Choe, and J.U. Jung. 2002. Herpesviral Protein Targets a Cellular WD Repeat Endosomal Protein to
Downregulate T Lymphocyte Receptor Expression. Immunity 17: 221-233.
Acknowledgements
We would like to thank the Henson School of Science & Technology at Salisbury
University and Glenda Gillaspy, Ph.D. at Virginia Tech in Blacksburg, VA for her helpful
comments throughout these procedures. Additionally, we would like to thank the USDA
for funding support.
Figure 1. Introducing Expression Vectors into
HeLa Cells. Cultured HeLa cells are plated in 6-well
culture dishes and transiently transfected with fusion
plasmids that express GFP. HeLa Monster and TransIT
reagent are used to increase the efficiency of DNA
uptake. After 48 hours, cells are then fixed with a 4%
paraformaldehyde solution, and stained with
fluorescent DAPI and Texas-Red phalloidin dyes.
The Procedure
Figure 2. Transient Transfection in HeLa Cells. (Above) DNA is stained with DAPI (A), a fluorescent molecule excited by UV light waves. Texas-Red phalloidin is introduced into the cell (B) and stains Actin microfilaments, while an
expression plasmid with a fluorescent gene product (GFP) is used to determine subcellular localization of the protein (C). Images are merged on top of one another (D) to illustrate relative locations of these fluorescent structures. Cells transfected
with the GFP expression plasmid show expression of the gene product throughout the cell (E and F); GFP localizes to the nucleus and cytoplasm. Previous localization studies have shown that GABARAP localizes to perinuclear vesicles (2) in the
cytoplasm which are few in number (G and H). GFP:Actin constructs were introduced to show colocalization patterns between the encoded fusion protein and the Actin microfilaments stained by Texas-Red (I and J). Fusion protein shows that
Myosin III extends to the filopodia tips (1) where it accumulates in bundles (K). Control cells, not transfected with GFP plasmid, show varying background levels of green fluorescence (L-N). GFP:p80 constructs (O-R) show trends similar to those
seen in control cells, indicating that perceived levels of expression may be background fluorescence.
AA
DNADNA
BB
ActinActin
CC
GFPGFP
DD
MergeMerge
DNA, Actin, Control
N
DNA, Actin, Control
M
DNA, Actin, Control
L
H
DNA, Actin, GFP:GABARAP
G
DNA, Actin, GFP:GABARAP
I
DNA, Actin, GFP:Actin
J
DNA, Actin, GFP:Actin
K
DNA, Actin, GFP:Myosin III
R
DNA, Actin, GFP:p80
Q
DNA, Actin, GFP:p80
P
DNA, Actin, GFP:p80
O
DNA, Actin, GFP:p80
F
DNA, Actin, GFP
E
DNA, Actin, GFP
Animal p80 Localizes to Endosomes
In animals, p80 functions in vesicle
trafficking and is involved in the
dowregulation of T-cell receptor
response (3). The functional pathway
recycles endosomal vesicles into and
out of the cell.
The predicted Arabidopsis gene At3g05090 may be the
plant p80 gene based on amino acid similarities; does plant
p80 protein also localize to endosomal vesicles?
The “80” Million Dollar Question
The Approach
Subcellular localization of the p80 protein can be
studied through fluorescence microscopy. Cultured
cells are transfected with GFP fusion genes and the
resulting fluorescent proteins are then analyzed
using microscopy.