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FYVE zinc-finger proteins in the plant model Arabidopsis thaliana: identification of PtdIns3P-binding residues by comparison of classic and variant FYVE domains.

机译:植物模型拟南芥中的FYVE锌指蛋白:通过比较经典和变异FYVE域来鉴定PtdIns3P结合残基。

摘要

Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)(1)(R/K)HHCR(6) (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins, named PRAF, consist of a pleckstrin homology (PH) domain, a regulator of chromosome condensation (RCC1) guanine nucleotide exchange factor repeat domain, and a variant FYVE domain containing an Asn residue and a Tyr residue at positions corresponding to the PtdIns3P-interacting His(4) and Arg(6) of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P(2), the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast, specificity for PtdIns3P was obtained by mutagenic conversion of the variant into a classic FYVE domain (Asn(4),Tyr(6)-->His(4),Arg(6)). Separate substitutions of the variant residues were not sufficient to impose preferential binding of PtdIns3P, suggesting a co-operative effect of these residues in binding. A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides.
机译:经典的FYVE锌指结构域识别磷酸肌醇信号PtdIns3P,并共享基本的(R / K)(1)(R / K)HHCR(6)(单字母氨基酸代码)共有序列。该结构域存在于拟南芥的预测的PtdIns3P 5激酶和脂肪酶中。其他称为PRAF的拟南芥蛋白由一个pleckstrin同源性(PH)域,一个染色体浓缩调节子(RCC1)鸟嘌呤核苷酸交换因子重复域和一个在对应于该位点的位置含有Asn残基和Tyr残基的变异FYVE域组成。 PtdIns3P交互的基本图案的His(4)和Arg(6)。在体外使用斑点印迹和脂质体结合测定法检查分离的PRAF结构域的磷脂结合能力。虽然PH域优先结合PtdIns(4,5)P(2),但变异FYVE域显示了弱的磷酸肌醇结合电荷依赖性。相反,对PtdIns3P的特异性是通过将变体诱变转化为经典的FYVE结构域而获得的(Asn(4),Tyr(6)-> His(4),Arg(6))。变异残基的单独取代不足以强加PtdIns3P的优先结合,表明这些残基在结合中具有协同作用。 PRAF的生物化学功能是通过其在Rab家族的一些小GTPases上催化鸟嘌呤核苷酸交换的能力来表明的,这使得可以讨论植物FYVE蛋白的生物学作用及其受磷酸肌醇的调控。

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