Functional analysis of Arabidopsis postprenylation CaaX processing enzymes and their function in subcellular protein targeting
Functional analysis of Arabidopsis postprenylation CaaX processing enzymes and their function in subcellular protein targeting
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DOI：
10.1104/pp.108.120477
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发表时间：
2008-09-01
期刊：
影响因子：
7.4
通讯作者：
Yalovsky, Shaul
中科院分区：
文献类型：
--
作者：
Bracha-Drori, Keren;Shichrur, Keren;Yalovsky, Shaul
异戊烯基化是一种蛋白质翻译后修饰，对发育过程和对脱落酸的反应至关重要。在异戊烯化之后，三个C-末端残基被蛋白水解去除，并且继而异戊烯基半胱氨酸的游离羧基被甲基化。蛋白水解和甲基化，统称为CaaX加工，由Ste 24内切蛋白酶或Rce 1内切蛋白酶和异戊二烯基半胱氨酸甲基转移酶（ICMT）催化。拟南芥（Arabidopsis thaliana）含有单个STE 24和RCE 1以及两个ICMT同源物。在这里，我们表明，在酵母（酿酒酵母）AtRCE 1促进a-交配因子的分泌和膜定位的ROP GT3。此外，AtSTE 24、AtRCE 1、AtICMTA和AtICMTB的绿色荧光蛋白融合蛋白共定位于内质网中，表明异戊二烯化蛋白到达该隔室，并且CaaX加工可能是亚细胞靶向所需的。AtICMTB能比AtICMTA更有效地加工酵母α-因子。序列和突变分析表明，较高的活性AtICMTB是由5个残基，这是保守的酵母Ste 14 p，人类ICMT，和AtICMTB之间，但不是在AtICMTA。实时定量逆转录聚合酶链反应和微阵列数据显示，AtICMTA的表达显着低于AtICMTB。AtICMTA无效突变体具有野生型表型，表明其功能是冗余的。然而，AtICMT RNAi株系具有扁化的花序茎，改变了趋化性，并且在没有茎伸长的情况下产生了多个芽。ICMT RNAi株系的表型与法尼基转移酶β亚基突变体增强对脱落酸的应答2相似，但更为微妙。总的来说，数据表明AtICMTB可能是主要的ICMT，甲基化调节异戊二烯化蛋白的活性。
Prenylation is a posttranslational protein modification essential for developmental processes and response to abscisic acid. Following prenylation, the three C-terminal residues are proteoliticaly removed and in turn the free carboxyl group of the isoprenyl cysteine is methylated. The proteolysis and methylation, collectively referred to as CaaX processing, are catalyzed by Ste24 endoprotease or Rce1 endoprotease and by an isoprenyl cysteine methyltransferase (ICMT). Arabidopsis (Arabidopsis thaliana) contains single STE24 and RCE1 and two ICMT homologs. Here we show that in yeast (Saccharomyces cerevisiae) AtRCE1 promoted a-mating factor secretion and membrane localization of a ROP GTPase. Furthermore, green fluorescent protein fusion proteins of AtSTE24, AtRCE1, AtICMTA, and AtICMTB are colocalized in the endoplasmic reticulum, indicating that prenylated proteins reach this compartment and that CaaX processing is likely required for subcellular targeting. AtICMTB can process yeast a-factor more efficiently than AtICMTA. Sequence and mutational analyses revealed that the higher activity AtICMTB is conferred by five residues, which are conserved between yeast Ste14p, human ICMT, and AtICMTB but not in AtICMTA. Quantitative real-time reverse transcription-polymerase chain reaction and microarray data show that AtICMTA expression is significantly lower compared to AtICMTB. AtICMTA null mutants have a wild-type phenotype, indicating that its function is redundant. However, AtICMT RNAi lines had fasciated inflorescence stems, altered phylotaxis, and developed multiple buds without stem elongation. The phenotype of the ICMT RNAi lines is similar to farnesyltransferase beta-subunit mutant enhanced response to abscisic acid2 but is more subtle. Collectively, the data suggest that AtICMTB is likely the major ICMT and that methylation modulates activity of prenylated proteins.