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Reversibility of Functional Transformation of Microbodies

微体功能转变的可逆性

基本信息

批准号：
07456053
负责人：
NISHIMURA Mikio
金额：
$ 4.93万
依托单位：
Okazaki National Rsearch Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1. In germinating fatty seedlings, microbodies are functionally transformed to leaf peroxisomes from glyoxysomes, and during greening to glyoxysomes from leaf peroxisomes during senesecence.Immunocytochemical studies have shown that glyoxysomes can exchange directly into leaf peroxisomes during greening and leaf peroxisomes are once again converted directly to glyoxysomes during senescence. To crarify the regulation underlying the microbody tranformation, transgenic Arabidopsis plants that expressed a fusion protein composed of the N-terminal region of and glyoxysomal citrate synthase (gCS) and beta-glucuromidase (GUS), were generated and their localization and processing were characterized by immunological and immunocytochemical methods. The fusion protein was transported into functionally different microbodies, such as glyoxysomes, leaf peroxisomes and unspecialized microbodies and was subsequently processed. These observations indicated that the transport of gCS is mediated by its a … More minoterminal presequence and that the transport system is functional in all plant microbodies. Therefore, it is unlikely that glyoxysomes and leaf peroxisomes possess different targeting machineries.2. Hydroxpyruvate reductase belongs to leaf-peroxisome specific enzymes and is accumulated in microbodies during greening. Two different hydroxypyruvate redutase (HPR1 and HPR2) are expressed in pumpkin. cDNA and genomic DNA cloning for HPR strongly suggests that HPR1 and HPR2 are produced by alternative splicing. Two different hydroxypryuvate reductase, HPR1 and HPR2, are shown to be localized in microbodies and in the cytosol respectively, suggesting the possibility that the microbody transition may be regulated by alternative splicing.3. Two proteins in glyoxysomal membranes with molecular masses of 31kDa and 28kDa were purified and characterized. The former was shown to be a novel ascorbate peroxidase. It was found that the amounts of these membrance proteins decreased during the microbody transition from glyoxysomes to leaf peroxisomes and that the large one was retained in leaf peroxisomes, whereas the small one could not be found in leaf peroxisomes after completion of the microbody transition. The results clearly showed that membrane proteins in glyoxysomes change dramatically during the microbody transition, as do the enzymes in the matrix. Less

1. 在发芽的脂肪幼苗中，微体在功能上由叶过氧化物酶体转化为叶过氧化物酶体，在衰老过程中由叶过氧化物酶体转化为叶过氧化物酶体。免疫细胞化学研究表明，在绿化过程中，glyoxysomes可以直接交换成叶片过氧化物酶体，而在衰老过程中，叶片过氧化物酶体再次直接转化为glyoxysomes。为了阐明微体转化的调控机制，我们构建了表达由和glyoxysomal citrate synthase （gCS）和β -glucuromidase （GUS）组成的融合蛋白的转基因拟南芥植株，并利用免疫学和免疫细胞化学方法对其定位和加工进行了表征。融合蛋白被转运到功能不同的微体中，如乙氧基小体、叶片过氧化物酶体和非特化微体，并随后被加工。这些结果表明，gCS的转运是由其a…More末端序列介导的，转运系统在所有植物微体中都起作用。因此，乙氧化酶体和叶过氧化物酶体不太可能具有不同的靶向机制。羟丙酮酸还原酶属于叶片过氧化物酶体特异性酶，在绿化过程中在微体中积累。南瓜中表达两种不同的羟丙酮酸还原酶（HPR1和HPR2）。HPR的cDNA和基因组DNA克隆有力地表明，HPR1和HPR2是通过选择性剪接产生的。两种不同的羟脯氨酸还原酶HPR1和HPR2分别定位于微体和细胞质中，这表明微体的过渡可能受到选择性剪接的调节。纯化并鉴定了两种分子质量分别为31kDa和28kDa的glyoxysomal membrane蛋白。前者被证明是一种新的抗坏血酸过氧化物酶。结果表明，这些膜蛋白在糖酶体向叶片过氧化物酶体转变的过程中数量减少，且大的膜蛋白保留在叶片过氧化物酶体中，而小的膜蛋白在完成微体转变后在叶片过氧化物酶体中已找不到。结果清楚地表明，在微体转变过程中，乙氧基体中的膜蛋白发生了巨大的变化，基质中的酶也发生了巨大的变化。少