Jacques Ismaele, Andrews Norma W, Huynh Chau
Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06510, USA.
Mol Biochem Parasitol. 2010 Mar;170(1):28-36. doi: 10.1016/j.molbiopara.2009.12.003. Epub 2009 Dec 16.
Leishmania amazonensis LIT1 was identified based on homology with IRT1, a ferrous iron transporter from Arabidopsis thaliana. Deltalit1L. amazonensis are defective in intracellular replication and lesion formation in vivo, a virulence phenotype attributed to defective intracellular iron acquisition. Here we functionally characterize LIT1, directly demonstrating that it functions as a ferrous iron membrane transporter from the ZIP family. Conserved residues in the predicted transmembrane domains II, IV, V and VII of LIT1 are essential for iron transport in yeast, including histidines that were proposed to function as metal ligands in ZIP transporters. LIT1 also contains two regions within the predicted intracellular loop that are not found in Arabidopsis IRT1. Deletion of region I inhibited LIT1 expression on the surface of Leishmania promastigotes. Deletion of region II did not interfere with LIT1 trafficking to the surface, but abolished its iron transport capacity when expressed in yeast. Mutagenesis revealed two motifs within region II, HGHQH and TPPRDM, that are independently required for iron transport by LIT1. D263 was identified as a key residue required for iron transport within the TPPRDM motif, while P260 and P261 were dispensable. Deletion of proline-rich regions within region I and between regions I and II did not affect iron transport in yeast, but in L. amazonensis were not able to rescue the intracellular growth of Deltalit1 parasites, or their ability to form lesions in mice. These results are consistent with a potential role of the unique intracellular loop of LIT1 in intracellular regulation by Leishmania-specific factors.
亚马逊利什曼原虫LIT1是基于与IRT1（拟南芥中的一种亚铁转运蛋白）的同源性而鉴定出来的。缺失lit1的亚马逊利什曼原虫在体内的细胞内复制和损伤形成方面存在缺陷，这种毒力表型归因于细胞内铁摄取缺陷。在这里，我们对LIT1进行了功能表征，直接证明它作为ZIP家族的亚铁膜转运蛋白发挥作用。LIT1预测的跨膜结构域II、IV、V和VII中的保守残基对于酵母中的铁转运至关重要，包括那些被认为在ZIP转运蛋白中作为金属配体发挥作用的组氨酸。LIT1在预测的细胞内环内还包含两个拟南芥IRT1中未发现的区域。区域I的缺失抑制了利什曼原虫前鞭毛体表面LIT1的表达。区域II的缺失不干扰LIT1向表面的运输，但当在酵母中表达时会消除其铁转运能力。诱变揭示了区域II内的两个基序，HGHQH和TPPRDM，它们是LIT1铁转运独立需要的。D263被确定为TPPRDM基序中铁转运所需的关键残基，而P260和P261是可有可无的。区域I内以及区域I和II之间富含脯氨酸区域的缺失不影响酵母中的铁转运，但在亚马逊利什曼原虫中无法挽救缺失lit1的寄生虫的细胞内生长或它们在小鼠中形成损伤的能力。这些结果与LIT1独特的细胞内环在利什曼原虫特异性因子的细胞内调节中的潜在作用一致。
