Lam-Yuk-Tseung Steven, Touret Nicolas, Grinstein Sergio, Gros Philippe
Department of Biochemistry, McGill Cancer Center and Center for Host Resistance, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Biochemistry. 2005 Sep 13;44(36):12149-59. doi: 10.1021/bi050911r.
Mutations in DMT1 (Nramp2 and Slc11a2) impair iron metabolism and cause microcytic anemia. DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron and is present at the plasma membrane and in recycling endosomes of most cells, where it is necessary for acquisition of transferrin-associated iron. The goal of this study was to identify signal(s) in the cytoplasmic segments of DMT1 responsible for its subcellular targeting and internalization from the plasma membrane into recycling endosomes. We introduced mutations in the amino terminus (DeltaNT), carboxyl terminus (DeltaCT), as well as in NPAY28-31, YSCF62-65, and YLLNT555-559 motifs of a DMT1 construct bearing an exofacial epitope tag, which allowed labeling of the transporter at the cell surface for kinetic studies. Mutants were stably expressed in LLC-PK1 kidney cells and were studied for transport activity, subcellular localization, cell-surface and recycling pool distribution, and internalization from the plasma membrane. Kinetic studies showed that carboxyl-terminus mutants (DeltaCT and DeltaYLLNT) had an increased fraction of the "recycling pool" that was expressed at the cell surface because of impaired internalization from the plasma membrane. Further cell-surface-labeling and immunofluorescence studies in intact cells showed that the DeltaYLLNT and DeltaCT mutants were targeted to the lysosomal compartment upon internalization. These results suggest that the major signal for internalization and recycling of DMT1 isoform II (-IRE/1B) resides in its carboxyl terminus and that removal of this signal leads to a default lysosomal targeting.
二价金属离子转运体1（DMT1，又称Nramp2和Slc11a2）发生突变会损害铁代谢并导致小细胞性贫血。DMT1在十二指肠刷状缘表达，在该处它控制膳食铁的摄取，并且存在于大多数细胞的质膜和再循环内体中，在这些部位它对于获取转铁蛋白相关铁是必需的。本研究的目的是确定DMT1胞质区段中负责其亚细胞靶向以及从质膜内化进入再循环内体的信号。我们在带有细胞外表面表位标签的DMT1构建体的氨基末端（DeltaNT）、羧基末端（DeltaCT）以及NPAY28 - 31、YSCF62 - 65和YLLNT555 - 559基序中引入突变，这使得能够在细胞表面标记该转运体以进行动力学研究。突变体在LLC - PK1肾细胞中稳定表达，并对其转运活性、亚细胞定位、细胞表面和再循环池分布以及从质膜的内化进行研究。动力学研究表明，羧基末端突变体（DeltaCT和DeltaYLLNT）由于从质膜内化受损，在细胞表面表达的“再循环池”部分增加。完整细胞中进一步的细胞表面标记和免疫荧光研究表明，DeltaYLLNT和DeltaCT突变体在内化后靶向溶酶体区室。这些结果表明，DMT1同工型II（-IRE/1B）内化和再循环的主要信号位于其羧基末端，去除该信号会导致默认的溶酶体靶向。
