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

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

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

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)

AtFHIT and AtHINT2 Putative PTS2 Signals

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.

Approach Using 35S-

radiolabeled protein

Methods Lane 1 and 2 are the

PCR amplified products of AtHINT2 and AtFHIT

SP6

T7

Inserted Gene

pCRII-TOPO PCR products were

then inserted into the pCRII-TOPO vector, the resulting plasmid was used to transform TOP10F’ E. coli

MethodsTransformed 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

MethodsClones 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

Results

TranslationTranslation

-Deg1

5+Deg1

5

-Deg1

5+Deg1

5

AtFHIT AtHINT2

*Expected molecular

mass= 20.4 kDa

Results

- + - + - +0

5

10

15

20

25

Substrate

Corr

ecte

d %

Pro

cess

ed

AtFHIT AtHINT2 AtASP3

Digital audioradiograph protease assay results

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

AcknowledgementsAli Dorchak

Dr. Laura OlsenThe Olsen Lab

Dr. Aaron WymanDr. Cherie DotsonDr. Ron Woodard

University of Michigan College of PharmacyThe National Science Foundation

Questions?