PDF(Pubmed)
Abstract：
Small GTPases switch between GDP- and GTP-bound states during cell signaling. The ADP-ribosylation factor (ARF) family of small GTPases is involved in vesicle trafficking. Although evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. We characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5 (TTN5; also known as HALLIMASCH, ARL2 and ARLC1) from Arabidopsis thaliana, and two TTN5 proteins with point mutants in conserved residues, TTN5T30N and TTN5Q70L, that were expected to be unable to perform nucleotide exchange and GTP hydrolysis, respectively. TTN5 exhibited very rapid intrinsic nucleotide exchange and remarkably low GTP hydrolysis activity, functioning as a non-classical small GTPase being likely present in a GTP-loaded active form. We analyzed signals from YFP-TTN5 and HA3-TTN5 by in situ immunolocalization in Arabidopsis seedlings and through use of a transient expression system. Colocalization with endomembrane markers and pharmacological treatments suggests that TTN5 can be present at the plasma membrane and that it dynamically associates with membranes of vesicles, Golgi stacks and multivesicular bodies. Although TTN5Q70L mirrored wild-type TTN5 behavior, the TTN5T30N mutant differed in some aspects. Hence, the unusual rapid nucleotide exchange activity of TTN5 is linked with its membrane dynamics, and TTN5 likely has a role in vesicle transport within the endomembrane system.
摘要：
小GTP酶在细胞信号传导中在GDP-和GTP-结合状态之间转换。ADP-核糖基化因子(ARF)家族参与囊泡运输。虽然进化上很保守，对植物中的ARF和ARF样GTP酶知之甚少。我们表征了拟南芥中必需的小ARF样GTPaseTITAN5/HALLIMASCH/ARL2/ARLC1的生化特性和细胞定位，包括两个保守点突变体，怀疑具有核苷酸交换和GTP水解的功能，TTN5T30N和TTN5Q70L。TTN5表现出非常快速的内在核苷酸交换和显着低的GTP水解活性，发挥非经典小GTP酶的功能，可能以GTP负载的活性形式存在。我们分析了拟南芥幼苗和瞬时表达系统中YFP-TTN5和HA3-TTN5的原位免疫定位信号。与内膜标记和药物治疗的共定位表明，TTN5可以存在于质膜上，并与囊泡膜动态相关，高尔基体和多囊体。虽然TTN5Q70L反映了野生型TTN5行为，TTN5T30N突变体在某些方面有所不同。因此，TTN5的异常快速核苷酸交换活性与膜动力学有关，可能与内膜系统中的囊泡运输有关。
