Topp Justin D, Gray Noah W, Gerard Robert D, Horazdovsky Bruce F
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
J Biol Chem. 2004 Jun 4;279(23):24612-23. doi: 10.1074/jbc.M313504200. Epub 2004 Mar 19.
ALS2 is the gene mutated in a recessive juvenile form of amyotrophic lateral sclerosis (ALS2). ALS2 encodes a large protein termed alsin, which contains a number of predicted cell signaling and protein trafficking sequence motifs. To gain insight into the overall function of alsin and to begin to evaluate its role in motor neuron maintenance, we examined the subcellular localization of alsin and the biochemical activities associated with its individual subdomains. We found that the Vps9p domain of alsin has Rab5 guanine nucleotide exchange activity. In addition, alsin interacted specifically with and acted as a guanine nucleotide exchange factor for Rac1. Immunofluorescence and fractionation experiments in both fibroblasts and neurons revealed that alsin is a cytosolic protein, with a significant portion associated with small, punctate membrane structures. Many of these membrane structures also contained Rab5 or Rac1. Upon overexpression of full-length alsin, the overexpressed material was largely cytosolic, indicating that the association with membrane structures could be saturated. We also found that alsin was present in membrane ruffles and lamellipodia. These data suggest that alsin is involved in membrane transport events, potentially linking endocytic processes and actin cytoskeleton remodeling.
ALS2是在一种隐性青少年型肌萎缩侧索硬化症(ALS2)中发生突变的基因。ALS2编码一种名为alsin的大蛋白,该蛋白包含许多预测的细胞信号传导和蛋白质运输序列基序。为了深入了解alsin的整体功能并开始评估其在运动神经元维持中的作用,我们研究了alsin的亚细胞定位及其各个亚结构域相关的生化活性。我们发现alsin的Vps9p结构域具有Rab5鸟嘌呤核苷酸交换活性。此外,alsin与Rac1特异性相互作用并作为其鸟嘌呤核苷酸交换因子发挥作用。在成纤维细胞和神经元中进行的免疫荧光和分级分离实验表明,alsin是一种胞质蛋白,其中很大一部分与小的点状膜结构相关。许多这些膜结构也含有Rab5或Rac1。全长alsin过表达后,过表达的物质主要存在于胞质中,这表明与膜结构的结合可能会饱和。我们还发现alsin存在于膜皱襞和片状伪足中。这些数据表明alsin参与膜运输事件,可能将内吞过程与肌动蛋白细胞骨架重塑联系起来。