Subramanian Shoba, Sijwali Puran S, Rosenthal Philip J
Department of Medicine, Division of Infectious Disease, University of California, San Francisco, San Francisco, California 94143, USA.
J Biol Chem. 2007 Aug 24;282(34):24961-9. doi: 10.1074/jbc.M703316200. Epub 2007 Jun 12.
The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 hydrolyze hemoglobin in an acidic food vacuole to provide amino acids for erythrocytic malaria parasites. Trafficking to the food vacuole has not been well characterized. To study trafficking of falcipains, which include large membrane-spanning prodomains, we utilized chimeras with portions of the proteases fused to green fluorescent protein. The prodomains of falcipain-2 and falcipain-3 were sufficient to target green fluorescent protein to the food vacuole. Using serial truncations, deletions, and point mutations, we showed that both a 20-amino acid stretch of the lumenal portion and a 10-amino acid stretch of the cytoplasmic portion of the falcipain-2 prodomain were required for efficient food vacuolar trafficking. Mutants with altered trafficking were arrested at the plasma membrane, implicating trafficking via this structure. Our results indicate that falcipains utilize a previously undescribed bipartite motif-dependent mechanism for targeting to a hydrolytic organelle, suggesting inhibition of this unique mechanism as a new means of antimalarial chemotherapy.
恶性疟原虫的半胱氨酸蛋白酶恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-3在酸性食物泡中水解血红蛋白，为红细胞内的疟原虫提供氨基酸。向食物泡的转运尚未得到很好的表征。为了研究包括大的跨膜前结构域的恶性疟原虫蛋白酶的转运，我们利用了蛋白酶部分与绿色荧光蛋白融合的嵌合体。恶性疟原虫蛋白酶-2和恶性疟原虫蛋白酶-3的前结构域足以将绿色荧光蛋白靶向到食物泡。通过连续截短、缺失和点突变，我们表明恶性疟原虫蛋白酶-2前结构域的腔部分的20个氨基酸片段和胞质部分的10个氨基酸片段对于有效的食物泡转运都是必需的。转运改变的突变体在质膜处停滞，表明通过该结构进行转运。我们的结果表明，恶性疟原虫蛋白酶利用一种以前未描述的二分基序依赖性机制靶向水解细胞器，这表明抑制这种独特机制作为抗疟化疗的新手段。
