拟南芥RabD2b蛋白氨基酸突变对定位和功能的影响

Rab蛋白4个保守的鸟嘌呤核苷酸结合结构域G1、G3、G4和G5共同参与了与GTP的结合及水解过程。将拟南芥(Arabidopsis thaliana)RabD2b(AtRabD2b)G4结构域的重要氨基酸位点天冬酰胺(asparagine, N)突变为异亮氨酸(isoleucine, I)(AtRabD2b^N121I]), 并研究了N121I突变对AtRabD2b亚细胞定位和功能的影响。结果表明, N121I突变使AtRabD2b由原来的高尔基体点状定位转变为高尔基体和细胞质弥散定位。AtRabD2b能够互补酿酒酵母(Saccharomyces cerevisiae)同源蛋白Ypt1突变产生的功能缺陷, 而AtRabD2b^N121I]仅能部分互补Ypt1的功能。AtRabD2b^N121I]转基因植株表现出矮化、丛生、不育和茎顶端坏死等多效性异常表型, 与AtRabD2b转基因植株出现的主茎异常抽出表型不同。上述结果表明, N121I突变不仅引起了AtRabD2b亚细胞定位的改变, 而且影响了其正常功能。

RabD2b Protein N121I Mutation Affects the Subcellular Localization and Functions in Arabidopsis thaliana

The four conserved guanine-base-binding motifs of Rab GTPase―G1, G3, G4 and G5―are involved in the binding and hydrolysis of GTP. We obtained the full-length coding sequence of the Arabidopsis thaliana RabD2b mutant allele AtRabD2b^N121I] within the conserved G4 motif by changing asparagine 121 to isoleucine and studied the effects of N121I mutation on the sublocalization and functions of AtRabD2b. The N121I mutation altered the specific localization of AtRabD2b from Golgi stacks to both Golgi and cytoplasm. AtRabD2b could completely complement the functional defect induced by the mutation of Saccharomyces cerevisiae Ypt1, which is homologous to AtRabD2b in yeast. However, At-RabD2b^N121I] only partly complemented the function of Ypt1. AtRabD2b^N121I] transgenic A. thaliana showed dwarf, bushy, sterile and necrotic phenotypes, which differed from that of AtRabD2b-overexpressing Arabidopsis with its abnormal main-inflorescence extension phenotype. The N121I mutation alters the subcellular localization of AtRabD2b and affects its normal functions.