| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
1) We first made recombinant yeast stains that express His-tagged Pex13p or Pex14p at their N- or C-terminus under the control of PEX14 promoter. The protein Complex containing these His-tag Pex14p (or Pex13p) was solubilized with Nonidet P-40 from organefle membrane and purified using Ni-affinity column. The duted fractions from it still contained a lot of contaminated proteins. Judging from the difference of behavior on Ni column, it was suggested that the protein complex containing mainly Pex14p might not include Pex13p. Next, we used a TAP (tandem affinity chromatography) system that contains sequential two-affinity chtomatography. We made recombinant yeast strains that express TAP-tag Pex14p. Using these systems we detected three protein bands of about 80, 63, 28kDa besides the tagged Pex14p in the purified fraction, suggesting that Pex14p might form a larger complex with these proteins. Currently, we are trying to identify them.
2) To establish in vitro import assay system we isolated alcohol oxidase (AO) and amine oxidase (AMD) genes of H. polymorpha using PCR, and subclonedlbsm into a vector (pCITE-2) for transcription. Currently, we are examining the conditions for in vitro import assay. On the other hand, to establish in vivo import assay system, we constructed fusion genes of GFP feat had PTS1(-SKL) at C-terminus or PTS2 of AMO at N-terminus. These fused genes were integrated into the AO promoter region of Δpex14 deletion mutant, and expressed under the control of AO promoter. When the wild type Pex14p was expressed in such cells using a vector containing zeocin gene as a marker, fluorescence spote of GEP were observed in the cells suggesting feat PTS-fused GFPs weie locafizied in peroxisomes. On the other hand, in the case of cells transformed with vector only, fluorescence was dispersed at the whole cytoplasm. These Δpex14 cells expressing PTS-fused GFP are useful to analyze the correlation between structure and function of Pex14p.
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