Sharer J D, Kahn R A
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050, USA.
J Biol Chem. 1999 Sep 24;274(39):27553-61. doi: 10.1074/jbc.274.39.27553.
ARF-like proteins (ARLs) comprise a functionally distinct group of incompletely characterized members in the ARF family of RAS-related GTPases. We took advantage of the GTP binding characteristics of human ARL2 to develop a specific, high affinity binding assay that allowed the purification of a novel ARL2-binding protein. A 19-kDa protein (BART, Binder of Arl Two) was identified and purified from bovine brain homogenate. BART binding is specific to ARL2.GTP with high affinity but does not interact with ARL2.GDP or activated ARF or RHO proteins. Based on peptide sequences of purified bovine BART, the human cDNA sequence was determined. The 489-base pair BART open reading frame encodes a novel 163-amino acid protein with a predicted molecular mass of 18,822 Da. Recombinant BART was found to bind ARL2.GTP in a manner indistinguishable from native BART. Northern and Western analyses indicated BART is expressed in all tissues sampled. The lack of detectable membrane association of ARL2 or BART upon activation of ARL2 is suggestive of actions quite distinct from those of the ARFs. The lack of ARL2 GTPase-activating protein activity in BART led us to conclude that the specific interaction with ARL2.GTP is most consistent with BART being the first identified ARL2-specific effector.
ARF样蛋白（ARLs）在RAS相关GTP酶的ARF家族中构成了一组功能独特但特征尚未完全明确的成员。我们利用人ARL2的GTP结合特性开发了一种特异性的高亲和力结合测定法，该方法可用于纯化一种新型的ARL2结合蛋白。从牛脑匀浆中鉴定并纯化出一种19 kDa的蛋白（BART，即Arl Two的结合蛋白）。BART与ARL2.GTP具有特异性的高亲和力结合，但不与ARL2.GDP或活化的ARF或RHO蛋白相互作用。基于纯化的牛BART的肽序列，确定了人cDNA序列。489个碱基对的BART开放阅读框编码一种新型的163个氨基酸的蛋白，预测分子量为18,822 Da。发现重组BART与ARL2.GTP的结合方式与天然BART无法区分。Northern和Western分析表明BART在所有采样组织中均有表达。ARL2激活后未检测到ARL2或BART与膜的结合，这表明其作用与ARFs的作用截然不同。BART缺乏ARL2 GTP酶激活蛋白活性，这使我们得出结论，与ARL2.GTP的特异性相互作用最符合BART是首个被鉴定出的ARL2特异性效应器这一情况。
