1. An Investigation of Two Putative
Peroxisomal Proteins: AtFHIT and
AtHINT2
Alyssa Castle1, Ali Dorchak2, Laura Olsen2
1Department of Molecular, Cellular, and Developmental
Biology, University of Michigan, Ann Arbor, Michigan

2. Peroxisomes
Molecular Biology of the Cell. 4th edition.
Alberts B, Johnson A, Lewis J, et al.
New York: Garland Science; 2002.
 Small membrane bound organelles
 Found in all eukaryotic cells
 Responsible for many metabolic
functions
 Catabolism of Long Chain Fatty
Acids
 Metabolism of H2O2
 Many peroxisomal disorders, usually
resulting in death in early stages of life
 Zellweger Syndrome
 X-Linked Adrenoleuknodystrophy
 D-Bifunctional Protein Deficiency

3. Peroxisomal Targeting Signals
 Peroxisomes lack their own genome
 Proteins are synthesized in the cytosol, then
transported into the peroxisome
 In order for proteins to be imported into
peroxisomes, they must have one or both PTS1
(Carboxyl-terminal) or PTS2 (Amino-terminal)
signals
 Like a zip code

4. Question/Hypothesis
 Do the AtFHIT and AtHINT2 encoding proteins
localize to the peroxisome?
 Hypothesize the proteins may localize in the
peroxisome because other proteins within this
‘family’ have been shown to be peroxisomal.
 HIT1 (PTS1), HIT2 (PTS2), HIT3 (PTS2)
(Reumann S et al. Plant Physiol. 2009;150:125-143)

5. AtFHIT and AtHINT2
 Putative PTS2 Signals

6. PTS2 Import Pathway
 Once the protein is translocated into the peroxisomal matrix, the
PTS2 signal at the amino terminus is cleaved by a protease
(DEG15) upon import into the peroxisome.

7. Approach
 Using 35S-
radiolabeled
protein

8. Methods
 Lane 1 and 2 are the PCR
amplified products of
AtHINT2 and AtFHIT
S
P
6
T
7
Inserted
Gene
pCRII-TOPO
 PCR products were
then inserted into the
pCRII-TOPO vector,
the resulting plasmid
was used to transform
TOP10F’ E. coli

9. Methods
Transformed cells were selected
using blue-white screening
Colonies were
selected and the
plasmid DNA
was mini
prepped
Restriction digests of the
mini prepped DNA were
done to drop inserts out
of the plasmid
Lanes 4 and 5 show a
successful drop of both
genes out of pCRII-
TOPO

10. Methods
Clones showing a successful drop
from the vector were sent for
sequencing
Maxi prepped clones and the
plasmid DNA was purified by
precipitation with PEG
Linearization and
purification
Transcription
Translation

11. Results
AtFHIT AtHINT2
*Expected
molecular mass=
20.4 kDa

12. Results
0
5
10
15
20
25
- + - + - +
Corrected%Processed
Substrate
AtFHIT AtHINT2 AtASP3
Digital audioradiograph protease
assay results

13. Conclusions
 In vitro experiments with AtFHIT and AtHINT2
show minimal processing
 Therefore are not very convincing whether they
are peroxisomal
 These results are not determinant of whether or
not the proteins are peroxisomal
 In vivo experiments would help to further
analyze whether these proteins are peroxisomal

14. Acknowledgements
Ali Dorchak
Dr. Laura Olsen
The Olsen Lab
Dr. Aaron Wyman
Dr. Cherie Dotson
Dr. Ron Woodard
University of Michigan College of Pharmacy
The National Science Foundation

15. Questions?