Natural resistance-associated macrophage protein 1 (NRAMP1) containing 548 amino acids (AA) and 12 transmembrane domains (TMDs) is localized in membranes of lysosomes. Our study aimed to investigate the targeting motifs of NRAMP1 by expressing GFP-tagged full-length and truncated NRAMP1 proteins and overlapping with the lysosomal marker Lamp1-RFP in Chinese hamster ovary (CHO) cells. The NH2-terminal amino acids 73-140 region including TMD2 was essential for NRAMP1 lysosomal targeting. The AA.263-334 region containing the tyrosine-based motif (327)YAPI(330) targeted NRAMP1 into lysosomes. Additionally, two internal signal peptides AA.451-483 and AA.489-522 were identified as lysosomal targeting motifs. Taken together, NRAMP1 consists of multiple targeting motifs for trafficking into lysosomes. (C) 2012 Elsevier Inc. All rights reserved.