| Research Abstract |
In this project, we analyzed the relation between the biochemical functions of peroxisomal membrane proteins (PMP) and the assembly of peroxisomes, and the sorting signal for PMP,dealing with the methlotrophic yeast Candida boidinii as a model organism.
By the analysis of regulation of the expression of peroxisomal matrix and membrane proteins, we conclude that the induction of PMP occurs before the matrix enzymes and that this is regulated mainly at the mRNA level. We cloned the PMP20, PMP30, PMP47 gene from C.boidinii, disrupted these genes, and analyzed growing activity of the disruption strains (pmp30DELTA and pmp47DELTA) on several carbon sources. Both strains could grow on oleate and D-alanine, but not on methanol ; these growth substrates require peroxisomal metabolism.
The results from the electron microscopic observation of pmp30DELTA showed that Pmp30 directly promotes peroxisome proliferation and affects organelle size and shape. And also we confirmed that Pmp30 is homologous
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to Pmp27 from Saccharomyces cerevisiae by genetic complementation experiments.
Methanol-induced cells of pmp47DELTA were investicated in detail. The activity of dihydroxyacetone synthase (DHAS), which is a methanol-induced peroxisome matrix enzyme, was not detected in pmp47DELTA. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body. Then, the PMP47 gene of two peroxisome-deficient mutants were disrupted. In these mutant strains, DHAS was enzymatically active and found in the cytoplasm. So we propose a mechanism that an unknown small molcule, which transports by Pmp47, is necessary for the folding or the translocation machinery of DHAS within peroxisomes.
Next, we analyzed the targeting sequence for peroxisomal integral membrane protein Pmp47, which spans the membrane six times. We demonstrated that the 20 amino acid loop, which predicted to face the matrix, is necessary and sufficient for sorting to peroxisomes. This is the first report of a sorting sequence, termed mPTS,for a peroxisomal integral membrane protein. Less
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