Adisa Akinola, Rug Melanie, Foley Michael, Tilley Leann
Department of Biochemistry, La Trobe University, Melbourne 3086, Vic, Australia.
Mol Biochem Parasitol. 2002 Aug 7;123(1):11-21. doi: 10.1016/s0166-6851(02)00117-2.
The mature human erythrocyte is a simple haemoglobin-containing cell with no internal organelles and no protein synthesis machinery. The malaria parasite invades this cell and develops inside a parasitophorous vacuole (PV). The parasite exports proteins into the erythrocyte to bring about extensive remodelling of its adopted cellular home. Plasmodial homologues of two COPII proteins, PfSar1p and PfSec31p, are exported to the erythrocyte cytosol where they appear to play a role in the trafficking of proteins across the erythrocyte cytoplasm [Eur. J. Cell Biol. 78 (1999) 453; J. Cell Sci. 114 (2001) 3377]. We have now characterised a homologue of the COPI protein, delta-COP. A recombinant protein corresponding to 90% of the Pfdelta-COP sequence was used to raise antibodies. The affinity-purified antiserum recognised a protein with an apparent M(r) of 58 x 10(3) on Western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. Pfdelta-COP was shown to be largely insoluble in non-ionic detergent, possibly suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of parasitised erythrocytes was used to show that, in contrast to the COPII proteins, Pfdelta-COP is located entirely within the parasite. The location of Pfdelta-COP partly overlaps that of the endoplasmic reticulum (ER)-located protein, PfERC, and partly that of the trans-Golgi-associated protein, PfRab6. Treatment of ring-stage plasmodium-infected erythrocytes with brefeldin A (BFA) inhibited development of the ER structure within the parasite cytosol and prevented the trafficking of the P. falciparum erythrocyte membrane protein-1, PfEMP1, to the erythrocyte cytosol. The Pfdelta-COP and PfSec31p populations each appear to be associated with the restricted ER structure in brefeldin-treated rings. When more mature stage parasites were treated with BFA, erythrocyte cytosol-located populations of parasite proteins were not reorganised, however, the overlap between Pfdelta-COP and PfERC in parasite cytosol was more complete suggesting a possible redistribution of the Golgi compartment into the ER. These data support the suggestion that both COPI and COPII proteins are involved in the trafficking of proteins within the parasite cytoplasm. However, only COPII proteins are exported to the erythrocyte cytosol to establish a vesicle-mediated protein trafficking pathway to the erythrocyte membrane.
成熟的人类红细胞是一种简单的含血红蛋白的细胞,没有内部细胞器,也没有蛋白质合成机制。疟原虫侵入这种细胞,并在一个寄生泡(PV)内发育。寄生虫将蛋白质输出到红细胞中,以对其寄生的细胞环境进行广泛重塑。两种COPII蛋白(PfSar1p和PfSec31p)的疟原虫同源物被输出到红细胞胞质溶胶中,它们似乎在蛋白质穿过红细胞细胞质的运输过程中发挥作用[《欧洲细胞生物学杂志》78 (1999) 453;《细胞科学杂志》114 (2001) 3377]。我们现在已经鉴定出了COP I蛋白δ-COP的一个同源物。一种对应于Pfδ-COP序列90%的重组蛋白被用来制备抗体。亲和纯化的抗血清在疟原虫感染的红细胞的蛋白质免疫印迹上识别出一种表观分子量为58×10³的蛋白质,但在未感染红细胞的印迹上未识别出。Pfδ-COP在非离子去污剂中基本不溶,这可能表明它与细胞骨架相连。利用共聚焦免疫荧光显微镜观察被寄生的红细胞,结果表明,与COPII蛋白不同,Pfδ-COP完全位于寄生虫内部。Pfδ-COP的定位部分与位于内质网(ER)的蛋白PfERC重叠,部分与反式高尔基体相关蛋白PfRab6重叠。用布雷菲德菌素A(BFA)处理环状期疟原虫感染的红细胞,可抑制寄生虫胞质溶胶中内质网结构的发育,并阻止恶性疟原虫红细胞膜蛋白-1(PfEMP1)运输到红细胞胞质溶胶中。在经布雷菲德菌素处理的环状体中,Pfδ-COP和PfSec31p群体似乎都与受限的内质网结构相关。当用BFA处理更成熟阶段的寄生虫时,寄生虫蛋白质位于红细胞胞质溶胶中的群体没有重新组织,然而,Pfδ-COP和PfERC在寄生虫胞质溶胶中的重叠更完全,这表明高尔基体区室可能重新分布到内质网中。这些数据支持了COP I和COPII蛋白都参与寄生虫细胞质内蛋白质运输的观点。然而,只有COPII蛋白被输出到红细胞胞质溶胶中,以建立一条囊泡介导的蛋白质运输途径至红细胞膜。