Khattab Ayman, Klinkert Mo-Quen
Department of Molecular Medicine, Bernhard-Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, Hamburg 20359, Germany.
Traffic. 2006 Dec;7(12):1654-65. doi: 10.1111/j.1600-0854.2006.00494.x. Epub 2006 Oct 2.
RIFINs are clonally variant antigens expressed in Plasmodium falciparum. Transfection and the green fluorescence protein (GFP) tagged either internally or C-terminally to the 3D7 PFI0050c RIFIN gene product were used to investigate protein localization, orientation and trafficking. Green fluorescence pattern emerging from live transfectant parasites expressing each of the RIFIN-GFP chimera was different. The internally GFP-tagged protein was exported to Maurer's clefts (MC) in the erythrocyte cytosol, whereas the C-terminally GFP-tagged full-length RIFIN chimera was not trafficked out of the parasite. Interestingly, when some RIFIN-specific C-terminal amino acid sequences were removed, the resulting truncated molecule reached the MC. Using anti-RIFIN and anti-GFP antibodies to probe both live and fixed transfectants, staining was confined to MC and was not detected on the erythrocyte surface, a location previously suggested for this protein family. From selective permeabilization experiments, the highly variable portion of the RIFIN-GFP-insertion chimera appeared to be exposed to the erythrocyte cytosol, presumably anchored in the MC membrane via the two transmembrane domains. Trafficking of both chimeras in young ring stages was sensitive to Brefeldin A (BFA), although older rings showed differential sensitivity to BFA.
RIFINs是恶性疟原虫中表达的克隆变异抗原。通过转染以及将绿色荧光蛋白（GFP）标记在3D7 PFI0050c RIFIN基因产物的内部或C末端，来研究蛋白质的定位、方向和运输。表达每种RIFIN-GFP嵌合体的活转染寄生虫产生的绿色荧光模式各不相同。内部标记GFP的蛋白质被转运至红细胞胞质溶胶中的毛雷尔氏小体（MC），而C末端标记GFP的全长RIFIN嵌合体则未被转运出寄生虫。有趣的是，当去除一些RIFIN特异性的C末端氨基酸序列时，产生的截短分子会到达MC。使用抗RIFIN和抗GFP抗体对活的和固定的转染体进行检测，染色局限于MC，在红细胞表面未检测到，而此前曾认为该蛋白家族位于该位置。从选择性透化实验来看，RIFIN-GFP插入嵌合体的高度可变部分似乎暴露于红细胞胞质溶胶中，推测是通过两个跨膜结构域锚定在MC膜上。两种嵌合体在年轻环状体阶段的运输对布雷菲德菌素A（BFA）敏感，尽管较老的环状体对BFA表现出不同的敏感性。
