National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India.
Plant Physiol. 2013 Mar;161(3):1141-57. doi: 10.1104/pp.112.206243. Epub 2013 Jan 2.
PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2') differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.
蛋白质 l-异天冬氨酰基甲基转移酶（PIMT）是一种广泛分布的蛋白质修复酶，可催化自发损伤蛋白质中异常 l-异天冬氨酰残基转化为正常天冬氨酰残基。该酶在植物中由两个不同的基因（PIMT1 和 PIMT2）编码，而在许多其他生物体中则不是这样。虽然已经阐明了 PIMT1 的生物学作用，但 PIMT2 基因在植物中的作用和意义尚未明确界定。在这里，我们从鹰嘴豆（Cicer arietinum）中分离出 PIMT2 基因（CaPIMT2），该基因在种子蛋白中异天冬氨酰残基显著增加，同时在人工老化条件下发芽活力降低。发现 CaPIMT2 基因高度分化，通过选择性剪接在 I 催化结构域区域编码两种可能的同工型（CaPIMT2 和 CaPIMT2'），相差两个氨基酸。与 CaPIMT1 不同，这两种同工型都具有独特的 56 个氨基酸氨基末端，表现出相似但不同的酶学特性。表达分析表明，CaPIMT2 受到应激和脱落酸的差异调控。稳定表达的绿色荧光蛋白融合 PIMT 蛋白的共聚焦可视化和细胞分级免疫印迹分析表明，除了质膜外，两种 CaPIMT2 同工型主要定位于核内，而 CaPIMT1 定位于细胞质中。值得注意的是，CaPIMT2 通过在拟南芥（Arabidopsis thaliana）中种子特异性表达，主要修复核蛋白中的异常异天冬氨酰残基，从而增强种子活力和寿命，而 CaPIMT1 通过主要修复胞质部分中的异常蛋白来增强种子活力和寿命。总之，我们的数据表明，CaPIMT2 很可能是通过基因复制进化而来的，随后发生亚功能化，专门修复核蛋白组。
