Progress 01/01/04 to 12/31/04
Outputs
We have made substantial progress towards our goals, but have some important experiments still to finish. The work with the PTS2 protein thiolase has been published. We showed that thiolase import into peroxisomes is dependent on its PTS2. Thiolase import is greatly stimulated by the addition of zinc to import reactions. In the course of this work, we found that the PTS1 and PTS2 protein import pathways interact synergistically. This surprising finding is the topic of a paper we are preparing to submit. Results from the second objective, to study the import/export of the PTS2 receptor are nearly finished and will be included in parts of two manuscripts. Our work with the putative processing protease is ongoing, as well. We have one manuscript almost ready to submit and have begun working with 2 additional peroxisomal proteases. Finally, we are bringing all of these goals together in our study of 2 peroxisomal proteins that appear to have both targeting signals. This
work is also ongoing.
Impacts
Asp3 is a new PTS2 protein, which increases the number of known substrates for the PTS2 import pathway. The mechanism of Pex7p function as a recycling receptor is important for an understanding of the biogenesis of the organelle. The potential export of the receptor is novel. This is the first identification of a PTS2 protein processing enzyme. Though it is not yet clear whether AtLon2p cleaves the PTS2 signal or whether it degrades the signal after cleavage, this is an important finding. We have recently begun to study two additional peroxisomal proteases. In addition, we have found two peroxisomal matrix proteins that appear to have both targeting signals. It will be very important to study the import parameters of these proteins to help us understand how the two pathways interact. Our work will provide significant new information about the pathways of protein import into plant peroxisomes - especially of oil seed plants. Seedlings from oil seed plants, such as
pumpkin and Brassica, contain agriculturally significant levels of lipids and have abundant glyoxysomes. Our work, with pumpkin glyoxysomes, will contribute to an understanding of the partitioning of fatty acids and lipids within oil seed cotyledons.
Publications
- Johnson T.L., and Olsen L.J. 2003. Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-Coenzyme A thiolase into glyoxysomes. Plant Physiol. 133:1991-1999.
- Goyer A., Johnson T.L., Olsen L.J., Collakova E., Shachar-Hill Y., Rhodes D., and Hanson A.D. 2004. Characterization and metabolic function of a peroxisomal sarcosine and pipecolate oxidase from Arabidopsis. J. Biol. Chem. 279:16947-16953.
- Johnson T.L., and Olsen L.J. 2005. Pex7p import and interaction with the PTS1 pathway in plant peroxisomes. To be submitted to The Plant Journal
- Johnson T.L., and Olsen L.J. 2005. Characterization of a Lon homolog found in plant peroxisomes. (manuscript in preparation)
- Olsen L.J., and Harrison-Lowe, N.J. 2005. Isolation of Plant Glyoxysomes. In Current Protocols in Cell Biology, Chapter 3 - Subcellular Fractionation. Edited by J.S. Bonifacino, J. Lippincott-Schwartz, M. Dasso, J. Harford, and K. Yamada. Invited chapter. (submitted)
- Uchida M, Trelease RN, Flynn CR, Lisenbee CS, Johnson TL, and Olsen LJ. 2005. In vivo localization of Pex5p and Pex7p to the cytosol of plant cells and in vitro evidence for import of AtPex5p into peroxisomes OR Localization of Pex5p and Pex7p in plant cells. (To be submitted to Planta.)
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Progress 10/01/02 to 09/30/03
Outputs
We have made progress on each of the three objectives from the proposal. First, we have continued work with the putative PTS2 protein Asp3. We have good evidence now that Asp3 follows the PTS2 protein import pathway. It appears to interact with the PTS2 protein receptor, Pex7p. We are just beginning studies on the role of the acid matrix on the import of PTS2 proteins. Second, we have shown that Pex7p is imported into peroxisomes, or at least to a protease-protected location on the membrane of the organelle. We cannot yet distinguish between these two possibilities. Finally, we have prepared a manuscript describing our results with a putative PTS2 protein processing enzyme, AtLon2p. AtLon2p specifically interacts with PTS2 proteins. It is a serine protease that is inhibited by zinc. AtLon2p is, itself, a PTS1 protein. Mutants in LON2 have decreased germination and slower growth than wild-type plants. Although good progress has been made on each objective, it has been
slower than desired due to the difficulty in hiring a good postdoc to work on the project.
Impacts
Asp3 is a new PTS2 protein, which increases the number of known substrates for the PTS2 import pathway. The mechanism of Pex7p function as a recycling receptor is important for an understanding of the biogenesis of the organelle. The potential export of the receptor is novel. This is the first identification of a PTS2 protein processing enzyme. Though it is not yet clear whether AtLon2p cleaves the PTS2 signal or whether it degrades the signal after cleavage, this is an important finding.
Publications
- Johnson T.L., and Olsen L.J. 2003. Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-Coenzyme A thiolase into glyoxysomes. Plant Physiol. 133:1991-1999.
- Goyer A., Johnson T.L., Olsen L.J., Collakova E., Shachar-Hill Y., Rhodes D., and Hanson A.D. 2004. Characterization and metabolic function of a peroxisoomal sarcosine and pipecolate oxidase from Arabidopsis. Submitted to J. Biol. Chem.
- Johnson T.L., and Olsen L.J. 2004. Pex7p import and interaction with the PTS1 pathway in plant peroxisomes. To be submitted to The Plant Journal
- Uchida M, Trelease RN, Flynn CR, Lisenbee CS, Johnson TL, and Olsen LJ. 2004. In vivo localization of Pex5p and Pex7p to the cytosol of plant cells and in vitro evidence for import of AtPex5p into peroxisomes OR Localization of Pex5p and Pex7p in plant cells. To be submitted to Planta.
- Johnson T.L., and Olsen L.J. 2004. Characterization of a Lon homolog found in plant peroxisomes. (manuscript in preparation)
- Brickner D.G., Crookes, W.J., and Olsen L.J. 2004. An acid matrix is required for peroxisome protein import into plant peroxisomes. (manuscript in preparation)
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