A novel molecular chaperone-like protein found in yeast peroxisomes

本项目旨在了解在酵母热带假丝酵母过氧化物酶体中发现的非特异性脂质转移蛋白（nsLTP或SCP2）的酵母同源物PXO-18的生理作用。到目前为止，还没有在过氧化物酶体中发现分子伴侣或应激蛋白。我们的研究结果表明PXP-18应该是一个可能的候选者。1)通过免疫电镜、亚细胞分离和蛋白质导入，PXP-18主要存在于过氧化物酶体基质中。PXP-18可保护过氧化物酶体酰基辅酶a氧化酶（ACO）在48或70ºC的温度下免于热失活。当在70℃加热15 min时，PXP-18和ACO亚基形成了一个接近化学计量的配合物，与它们的初始比例无关。在30℃的温度下，游离的ACO从分离的配合物中自发释放出来。PXP-18在70℃时二级结构发生可逆变化，并随时间和温度的变化形成同型二聚体。二聚体PXP-18可以与部分变性的八聚ACOs结合形成近化学计量络合物，阻止其进一步变性。2)对秀丽隐杆线虫（Caenorhabditis elegans）进行cDNA克隆，发现一种与硫酶-SCP2高度相似但缺乏SCP2片段的蛋白（TLP）；硫酶-SCP2由哺乳动物SCP2基因的较长mRNA编码，具有3-氧酰基辅酶a硫酶活性。TLP和PXP-18似乎代表了硫酶- scp2的进化起源。PXP-18可能像泛素一样，促进融合硫硫酶与假定的β -氧化复合物的结合。泛素，一种应激蛋白，也以融合形式与核糖体蛋白表达，并促进其结合到核糖体中。

This project aimed at understanding the physiological role of a yeast homologue of nonspecific lipid-transfer protein (nsLTP or SCP2) , which we found in peroxisomes of the yeast Candida tropicalis and named PXO-18. No molecular chaperone or stress protein has been found in peroxisomes thus far. Our results suggest that PXP-18 should be a possible candidate for it.1) PXP-18 was shown to be present predominantly in the matrix of peroxisomes by means of immunoeLectron microscopy, subcellular fractionation, and by importing the protein. PXP-18 protected peroxisomal acyl-CoA oxidase (ACO) from thermal inactivation at 48ﾟC or 70ﾟC.When heated at 70ﾟC for 15 min, PXP-18 and ACO subunit formed a nearstoichiometric complex, independent of their initial ratio. Free ACO was released from the isolated complex spontaneously at 30ﾟC.PXP-18 changed its secondary structure reversibly at 70ﾟC,and formed homodimers in a manner dependent on time and temperature. The dimeric PXP-18 may bind to octameric ACOs denatured partially to form the nearstoichiometric complex, preventing their further denaturation.2) A cDNA clone of the nematode Caenorhabditis elegans revealed a protein (TLP) that is highly similar to thiolase-SCP2 but lacks SCP2 moiety ; thiolase-SCP2 is encoded by the longer mRNA from the mammalian SCP2 gene and shows 3-oxoacyl-CoA thiolase activity. TLP and PXP-18 seems to represent the evolutionary origins of thiolase-SCP2. PXP-18 may facilitate the incorporation of the fused thiolase into a putative beta-oxidation complex, as ubiquitin does. Ubiquitin, a stress protein, is also expressed as a fused form with ribosomal proteins and facilitates their incorporation into ribosomes.