Gruenheid S, Pinner E, Desjardins M, Gros P
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
J Exp Med. 1997 Feb 17;185(4):717-30. doi: 10.1084/jem.185.4.717.
The Nramp1 (natural-resistance-associated macrophage protein 1) locus (Bcg, Ity, Lsh) controls the innate resistance or susceptibility of mice to infection with a group of unrelated intracellular parasites which includes Salmonella, Leishmania, and Mycobacterium. Nramp1 is expressed exclusively in professional phagocytes and encodes an integral membrane protein that shares structural characteristics with ion channels and transporters. Its function and mechanism of action remain unknown. The intracellular localization of the Nramp1 protein was analyzed in control 129/sv and mutant Nramp1-/- macrophages by immunofluorescence and confocal microscopy and by biochemical fractionation. In colocalization studies with a specific anti-Nramp1 antiserum and a panel of control antibodies directed against known cellular structures, Nramp1 was found not to be expressed at the plasma membrane but rather localized to the late endocytic compartments (late endosome/lysosome) of resting macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-positive compartment. Double immunofluorescence studies and direct purification of latex bead-containing phagosomes demonstrated that upon phagocytosis, Nramp1 is recruited to the membrane of the phagosome and remains associated with this structure during its maturation to phagolysosome. After phagocytosis, Nramp1 is acquired by the phagosomal membrane with time kinetics similar to Lamp1, but clearly distinct from those of the early endosomal marker Rab5. The targeting of Nramp1 from endocytic vesicles to the phagosomal membrane supports the hypothesis that Nramp1 controls the replication of intracellular parasites by altering the intravacuolar environment of the microbe-containing phagosome.
Nramp1（天然抗性相关巨噬细胞蛋白1）基因座（Bcg、Ity、Lsh）控制小鼠对一组不相关细胞内寄生虫感染的先天抗性或易感性，这些寄生虫包括沙门氏菌、利什曼原虫和分枝杆菌。Nramp1仅在专职吞噬细胞中表达，编码一种与离子通道和转运蛋白具有结构特征的整合膜蛋白。其功能和作用机制尚不清楚。通过免疫荧光、共聚焦显微镜以及生化分级分离法，对对照129/sv小鼠和Nramp1基因敲除突变体巨噬细胞中Nramp1蛋白的细胞内定位进行了分析。在与特异性抗Nramp1抗血清以及一组针对已知细胞结构的对照抗体进行的共定位研究中，发现Nramp1不在质膜上表达，而是定位于静息巨噬细胞晚期内吞区室（晚期内体/溶酶体）中Lamp1（溶酶体相关膜蛋白1）阳性区室。双重免疫荧光研究以及对含乳胶珠吞噬体的直接纯化表明，吞噬作用发生时，Nramp1被募集到吞噬体膜上，并在其成熟为吞噬溶酶体的过程中一直与该结构相关联。吞噬作用后，Nramp1随时间动力学被吞噬体膜获取，其时间动力学与Lamp1相似，但明显不同于早期内体标志物Rab5。Nramp1从内吞小泡靶向至吞噬体膜支持了这样一种假说，即Nramp1通过改变含微生物吞噬体的泡内环境来控制细胞内寄生虫的复制。
