ARL4, an ARF-like protein that is developmentally regulated and localized to nuclei and nucleoli
ARL4, an ARF-like protein that is developmentally regulated and localized to nuclei and nucleoli
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DOI：
10.1074/jbc.m002470200
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发表时间：
2000-12-01
影响因子：
4.8
通讯作者：
Lee, FJS
中科院分区：
文献类型：
--
作者：
Lin, CY;Huang, PH;Lee, FJS
ADP 核糖基化因子 (ARF) 与 20-kDa 鸟嘌呤核苷酸结合蛋白高度保守，通过仅部分了解的机制参与胞吐和胞吞囊泡转运途径。尽管已知几种 ARF 样蛋白 (ARL)，但它们的生物学功能仍不清楚。为了表征其分子特性，我们克隆了小鼠和人类 ARL4（mARL4 和 hARL4）cDNA。小鼠 ARL4 mRNA 在胚胎发育过程中的出现与体节的顺序形成和脑区室的建立在时间上一致。使用ARL4特异性抗体进行免疫荧光显微镜观察，我们观察到培养的支持细胞和神经母细胞瘤细胞中的内源性mARL4主要集中在细胞核中。当在 COS7 细胞中表达时，预计与 GDP 结合存在的 ARL4-T34N 突变体集中在核仁中。酵母双杂交筛选和体外蛋白质相互作用测定表明，hARL4 通过其 C 末端 NLS 区域与 importin-alpha 相互作用，并且这种相互作用不依赖于核苷酸。与 ARL2 和 -3 一样，重组 hARL4 不会增强霍乱毒素催化的自动 ADP 核糖基化。其鸟苷 5'-O-（硫代三磷酸）的结合被磷脂和去垢剂修饰，并且 hARL4 的 N 末端与 ARF 一样被肉豆蔻酰化。我们的研究结果表明，ARL4 以其独特的核/核仁定位和发育表达模式，可能在胚胎发育期间和成人精子发生早期阶段的神经发生和体节发生中发挥独特的作用。
ADP-ribosylation factors (ARFs) are highly conserved similar to 20-kDa guanine nucleotide-binding proteins that participate in both exocytic and endocytic vesicular transport pathways via mechanisms that are only partially understood. Although several ARF-like proteins (ARLs) are known, their biological functions remain unclear. To characterize its molecular properties, we cloned mouse and human ARL4 (mARL4 and hARL4) cDNA. The appearance of mouse ARL4 mRNA during embryonic development coincided temporally with the sequential formation of somites and the establishment of brain compartmentation. Using ARL4-specific antibody for immunofluorescence microscopy, we observed that endogenous mARL4 in cultured Sertoli and neuroblastoma cells was mainly concentrated in nuclei. When expressed in COS7 cells, ARL4-T34N mutant, predicted to exist with GDP bound, was concentrated in nucleoli. Yeast two-hybrid screening and in vitro protein-interaction assays showed that hARL4 interacted with importin-alpha through its C-terminal NLS region and that the interaction was not nucleotide-dependent. Like ARL2 and -3, recombinant hARL4 did not enhance cholera toxin-catalyzed auto-ADP-ribosylation. Its binding of guanosine 5'-O-(thiotriphosphate) was modified by phospholipid and detergent, and the N terminus of hARL4, like that of ARF, was myristoylated. Our findings suggest that ARL4, with its distinctive nuclear/nucleolar localization and pattern of developmental expression, may play a unique role(s) in neurogenesis and somitogenesis during embryonic development and in the early stages of spermatogenesis in adults.