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内容記述 |
In higher plants, it have been clarified that glyoxysomes contain enzymes for the fatty acid β-oxidation cycle and the glyoxylate cycle whereas leaf peroxisomes contain enzymes of the photorespiration pathway. Recently, the complete genome sequences of Arabiclopsis were determined, and 25,498 genes were annotated in the genome. It is deduced from the genome search that 281 genes for proteins with peroxisomal targeting signals (PTS1 and PTS2) or proteins related to the functions of peroxisomes are present in the Arabidopsis genome. Also, recent data indicate that peroxisomal membranes are impermeable to small solutes, although it has not been identified the specific transporters for them. These data suggests that peroxisomes have novel proteins and they have novel functions. He performed the following experiments to clarify the novel functions of peroxisomes.<br /> In Chapter 2, to clarify the peroxisomal membrane proteins (PMPs), he characterized one of the major PMPs, PMP38. The deduced amino acid sequence for its cDNA in Arabidopsis had high similarity with those of Homo sapiens PMP34 and Candida boidinii PMP47 known as homologues of mitochondrial ATP/ADP carrier protein. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. Peroxisomes has been thought to be transported ATP into peroxisomes for various reactions, because ATP-synthesis pathways have not been found in peroxisomes. Also, the amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein, PMP38 in glyoxysomes changes dramatically during transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal proteins, especially proteins of the fatty acid β-oxidation cycle, that are localized in the matrix of glyoxysomes.<br /> In Chapter 3, to better understand the functions of leaf peroxisomes, he established a method for isolation of leaf peroxisomes from green cotyledons. And then, he performed proteomic analysis combined with 2-D get electrophoresis and MALDI-TOF MS. He analyzed 53 proteins and identified 29 proteins. Among them, 19 novel proteins were identified except the known leaf peroxisomal proteins, suggesting that leaf peroxisomes contain novel proteins and functions.<br /> In Chapter 4, to clarify the functions of glyoxysomes, he developed the method that he established in Chapter 3 to glyoxysomal proteomic analysis, and identified 19 proteins including 13 novel proteins. Especially, he analyzed serine/threonine protein kinase-like protein identified as one of the novel peroxisomal proteins in detail. He designated this protein peroxisomal protein kinase 1 (PPK1). It has been never proved that protein kinases are localized in peroxisomes. Also, it is a crucial point to confirm the localization of novel identified proteins for proteomic analysis of organelles. The analysis of suborganellar localization and protease sensitivity revealed that PPK1 is a glyoxysomal membrane protein and the putative protein kinase domain is located inside of in glyoxysomes in Chapter 5. He measured the phosphorylation activity of endogenous PPK1 in vitro using glyoxysomes isolated from etiolated pumpkin cotyledons. He revealed that the 40 kDa protein was phosphorylated by PPK1 in glyoxysomes, supporting that peroxisome have sophisticated phosphorylation systems. |
要旨・審査要旨 / Abstract, Screening Result (238.0 kB)
