Thelen J J, Mekhedov S, Ohlrogge J B
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824, USA.
Plant Physiol. 2001 Apr;125(4):2016-28. doi: 10.1104/pp.125.4.2016.
Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits. One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single gene in Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identity to AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms were expressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted with purified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea (Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylated polypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 protein was present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarily expressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcript reflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereas AtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected in developing seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereas developing seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate that oilseed plants in the family Brassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upon genome complexity.
大多数植物中的质体乙酰辅酶A羧化酶是一种由四个不同亚基组成的多酶复合体。其中一个亚基，生物素羧基载体蛋白（BCCP），先前被认为在拟南芥中由单个基因编码。我们在此报告并鉴定了第二个拟南芥BCCP（AtBCCP2）cDNA，它与AtBCCP1的氨基酸同一性为42%，与一类油菜（甘蓝型油菜）BCCP的同一性为75%。两种拟南芥BCCP同工型都在大肠杆菌中表达，并且在用纯化的重组拟南芥生物素羧化酶重构时被发现发生了生物素化并支持羧化活性。体外翻译的AtBCCP2能够导入豌豆（豌豆）叶绿体并加工成25-kD的多肽。拟南芥种子提取物含有35-kD和25-kD的生物素化多肽，分别与重组AtBCCP1和2的质量一致。AtBCCP1蛋白存在于根、叶、花、角果和种子的发育组织中，而AtBCCP2蛋白主要在开花后7至10天的种子中表达，其水平比AtBCCP1低约2倍。AtBCCP1转录本反映了所有发育器官中的这些蛋白质表达谱，在14天龄的叶片和角果中最高，而AtBCCP2转录本存在于花和角果中。在蛋白质印迹中，在油菜发育的种子中检测到四种不同的BCCP同工型。其中，在未成熟叶片和发育的种子中检测到一种35-kD的BCCP，而发育的种子中还含有在开花后21天高度表达的22-kD、25-kD和37-kD同工型。这些数据表明，十字花科的油料植物根据基因组复杂性至少含有一到三种种子上调的BCCP同工型。
