Burkhead Jason L, Abdel-Ghany Salah E, Morrill Jonathan M, Pilon-Smits Elizabeth A H, Pilon Marinus
Biology Department, Colorado State University, Room E 416, Fort Collins, CO 80523, USA.
Plant J. 2003 Jun;34(6):856-67. doi: 10.1046/j.1365-313x.2003.01769.x.
The Arabidopsis thaliana CUTA gene encodes a 182-amino-acid-long putative precursor of a chloroplast protein with high sequence similarity to evolutionarily conserved prokaryotic proteins implicated in copper tolerance. Northern analysis indicates that AtCUTA mRNA is expressed in all major tissue types. Analysis of cDNA clones and RT-PCR with total mRNA revealed alternative splicing of AtCUTA by retention of an intron. The intron-containing mRNA encodes a truncated 156-amino-acid protein as a result of stop codons in the included intron. The sequence of AtCutAp encoded by the fully spliced transcript suggests that the precursor consists of three domains: an N-terminal chloroplast transit sequence of 70 residues, followed by a domain with prokaryotic signal-sequence-like characteristics and finally the most conserved C-terminal domain. The N-terminal chloroplast transit sequence was functional to route a passenger protein into isolated pea chloroplasts with possible sorting to the envelope. Chloroplast localization was confirmed by Western blot analysis of isolated and fractionated chloroplasts. Recombinant AtCutA protein was expressed in Escherichia coli without the N-terminal 70-amino-acid chloroplast transit sequence. This recombinant AtCutAp was routed to the bacterial periplasm of E. coli. Purified recombinant AtCutAp is tetrameric and selectively binds Cu(II) ions with an affinity comparable to that reported for mammalian prion proteins.
拟南芥CUTA基因编码一种182个氨基酸长的叶绿体蛋白假定前体，与参与铜耐受性的进化保守原核蛋白具有高度序列相似性。Northern分析表明AtCUTA mRNA在所有主要组织类型中均有表达。对cDNA克隆的分析以及用总mRNA进行的RT-PCR显示，AtCUTA通过保留一个内含子而发生可变剪接。由于包含的内含子中有终止密码子，含内含子的mRNA编码一种截短的156个氨基酸的蛋白质。由完全剪接的转录本编码的AtCutAp序列表明，前体由三个结构域组成：一个70个残基的N端叶绿体转运序列，接着是一个具有原核信号序列样特征的结构域，最后是最保守的C端结构域。N端叶绿体转运序列可将一个报告蛋白转运到分离的豌豆叶绿体中，并可能分选到包膜上。通过对分离和分级分离的叶绿体进行蛋白质印迹分析证实了叶绿体定位。重组AtCutA蛋白在大肠杆菌中表达，没有N端70个氨基酸的叶绿体转运序列。这种重组AtCutAp被转运到大肠杆菌的细菌周质中。纯化的重组AtCutAp是四聚体，能选择性结合Cu(II)离子，其亲和力与报道的哺乳动物朊病毒蛋白相当。
