This document summarizes research on the Arabidopsis thaliana protein At3g05090, which contains WD repeat domains like the animal endosomal protein p80. Yeast two-hybrid screens identified four proteins that interact with At3g05090, including one involved in auxin transport. When fused to GFP, At3g05090 localized to the cytoplasm and vesicles in human HeLa cells, similarly to endosomal markers. Future work will further characterize At3g05090's role in endosomal trafficking and auxin signaling by generating knockout plants and studying subcellular localization.

1. 1
Expression and localization of the Arabidopsis
thaliana p80 protein in a human cancer cell line
Joseph S. Danner, Amanda Ely, Philip Ferralli, Francis
Owusu, and F. Les Erickson
Department of Biological Sciences,
Salisbury University
Salisbury, MD 21801
At3g05090 encodes a protein having a WD
repeat domain
• WD-repeats are stretches of
amino acids that end in Trp-Asp
and are responsible for Atp80’s
propeller like structure.
• These stretches consist of
approximately 40 amino acids.
• The WD repeat domain is
responsible for protein-protein
interactions.
• The Arabidopsis genome
encodes for 237 potential
proteins consisting of ≥4 WD
motifs.
The G protein beta subunit
contains 7 WD-repeat motifs
At3G05090
• Gene has no ascribed function to date.
• Gene product consists of 7 WD motifs
• Single gene in Arabidopsis and in rice.
• Resembles the p80 endosomal protein in animals.
82 kDaN C?WDWD WD WD WD WD WD
p80 Function in Animals
Immunity, Vol. 17, 221–233, August, 2002
Herpesviral Protein Targets a Cellular WD Repeat Endosomal
Protein to Downregulate T Lymphocyte Receptor Expression
Junsoo Park, Bok-Soo Lee, Joong-Kook Choi, Robert E. Means, Joonho Choe, and Jae U. Jung
Department of Microbiology and Molecular Genetics, Harvard Medical School
Department of Biological Sciences, Korea Advanced Institute of Science and Technology
Animal p80 endosomal protein
N CWDWD WD WD WD WD WD
Endosomal membrane
targeting
Herpesvirus Tip
interaction

2. 2
At3g05090 protein is 35% identical and 50%
similar to animal endosomal protein
Yeast 2-hybrid screenings for isolation of
Atp80 interaction proteins
0
2
4
6
8
10
12
14
16
18
20
Empty
Prey
30 33 42 50
Prey Genes
β-GalactosidaseUnits
BAIT
N C?WDWD WD WD WD WD WD
•The C-terminal domain was
used as BAIT and the cDNA
library (Arabidopsis) was
screened for positive
interactions.
•>2 million clones were
screened. Four produced
positive interactions with
Atp80.
•PREY plasmids were isolated
from positive colonies,
retested, and sequenced.
Atp80 Interacting Proteins
Annexin I, a Ca
2+
-dependent membrane
binding proteins involved in membrane
trafficking and stress responses.
At1g3572042
PINOID Binding Protein 1; PINOID kinase
regulates auxin transport via endosomal
trafficking of PIN1
At5g5449050
Unknown function; no homologiesAt5g4205033
Unknown function; Drosophila homolog
interacts with Hrs, a key regulator of vesicular
transport
At5g5120030
DescriptionArabidopsis genePREY #
Localization of Atp80 in HeLa cells
Protein “X”EGFP
Filopodia cell extensionsEGFP:Myosin*
CytoplasmEGFP:Myosin (mutant)*
Endosomal vesicles?EGFP:p80
Endosomal vesiclesEGFP:Rab*
Perinuclear vesiclesEGFP:GABARAP
Actin cytoskeletonEGFP:Actin
Cytoplasm and nucleusEGFP
Subcellular locationGene Construct
HeLa cells were transfected with
various gene constructs to
establish the localization scheme
of Atp80.
Side by side comparison of our
marker proteins with the
EGFP:p80 fusion plasmid show
that Atp80 is expressed
independently of the nucleus.
More tests must be performed. * construct not shown
Why HeLa Cells?
HeLa cells were ideal for our research because genes can easily be introduced for
transient or stable expression.
Many molecular tools are available for HeLa cells, making them an attractive
alternative to plant cells. Additionally, HeLa cells can be easily grown and
maintained in culture where it would be difficult to maintain plant cultures.

3. 3
Localization of Atp80 in HeLa cells
Transfection Process
DAY 1
• HeLa cells were plated in 6 well dishes and incubated for 24 hours.
DAY 2
• Cells were transfected using our gene constructs and HeLa TransIT
reagent.
• HeLa Monster reagent was used to increase the efficiency of our
transfections.
DAY 3
• Cells were fixed using 4% Paraformaldehyde solution and mounted
using an antifade reagent with DAPI stain. The DAPI stain will allow
the nuclei to appear blue when using fluorescence microscopy.
Localization of Atp80 in HeLa cells
GFP: GABARAPGFP
GFP: p80GFP: Actin
Auxin Signaling
• The auxin efflux transporter,
PIN 1, is regulated via the
endosomal cycle.
• In animals, T-cell receptors are
regulated in a similar manner
and p80 is introduced in
endosomal trafficking.
• p80 interacts with PBP 1
through the C-terminal domain
as shown in the results obtained
from the yeast 2-hybrid
screenings.
PIN 1
p80?
PBP 1
PINOID
Intracellular Matrix
Conclusions and Future Experiments
• More experiments must be done to conclude whether or not p80 is
endosomally localized although localization experiments have shown
promising results.
• An endosomal marker, such as Rab, must be used to indefinitely
conclude whether or not p80 is endosomally localized.
• We have not had enough time to take pictures so any conclusions
beyond p80 being cytoplasmic and not nuclear must still be determined.
Future projects….
•Atp80 35S, gene knock-out, and RNAi plants; look for auxin phenotypes
•Atp80:GFP plants for subcellular localization studies
•Two hybrid screens using the WD-repeat domain of Atp80.

4. 4
Acknowledgements
• Amanda Ely (Set-up yeast 2-hybrid experiments)
• Philip Ferralli (Performed β-Gal assays)
• Francis Owusu (Set-up yeast 2-hybrid experiments)
• Dr. Les Erickson, SU, advisor
• Dr. Glenda Gillaspy, Virginia Tech, collaborator
Funding
• Salisbury University, Henson Undergraduate Research Grants
• USDA Higher Education Challenge Grant