Daubenberger Claudia A, Tisdale Ellen J, Curcic Marija, Diaz Diana, Silvie Olivier, Mazier Dominique, Eling Wijnand, Bohrmann Bernd, Matile Hugues, Pluschke Gerd
Molecular Immunology, Swiss Tropical Institute, Socinstr. 57, CH-4002 Basel, Switzerland.
Biol Chem. 2003 Aug;384(8):1227-37. doi: 10.1515/BC.2003.135.
Spatial and temporal distribution of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (pfGAPDH) and aldolase (pfAldolase) of Plasmodium falciparum were investigated using specific mAbs and indirect immunofluorescence analysis (IFA). Both glycolytic enzymes were co-localized during ring and trophozoite stages of both liver and asexual blood stage parasites. During schizogony, pfGAPDH became associated with the periphery of the parasites and eventually accumulated in the apical region of merozoites, while pfAldolase showed no segregation. Subcellular fractionation experiments demonstrated that pfGAPDH was found in both the membrane-containing pellet and the supernatant fraction of parasite lysates. In contrast, pfAldolase was only found in the supernatant fraction. A quantitative binding assay showed that pfGAPDH could be recruited to HeLa cell microsomal membranes in response to mammalian GTPase Rab2, indicating that Rab2-dependent recruitment of cytosolic components to membranes is conserved in evolution. Two overlapping fragments of pfGAPDH (residues 1-192 and 133-337) were evaluated in the microsomal binding assay. We found that the N'-terminal fragment competitively inhibited Rab2-stimulated pfGAPDH recruitment. Thus, the domain mediating the evolutionarily conserved Rab2-dependent membrane recruitment is located in the N'-terminus of GAPDH. Together, these results suggest that pfGAPDH exerts non-glycolytic function(s) in P. falciparum, possibly including a role in vesicular transport and biogenesis of apical organelles.
利用特异性单克隆抗体和间接免疫荧光分析（IFA），研究了恶性疟原虫糖酵解酶甘油醛-3-磷酸脱氢酶（pfGAPDH）和醛缩酶（pfAldolase）的时空分布。在肝脏和无性血液阶段寄生虫的环状体和滋养体阶段，这两种糖酵解酶都共定位。在裂殖生殖过程中，pfGAPDH与寄生虫的外周相关联，并最终积聚在裂殖子的顶端区域，而pfAldolase没有分离。亚细胞分级分离实验表明，pfGAPDH存在于寄生虫裂解物的含膜沉淀和上清液部分中。相比之下，pfAldolase仅存在于上清液部分中。定量结合试验表明，pfGAPDH可响应哺乳动物GTP酶Rab2被募集到HeLa细胞微粒体膜上，这表明Rab2依赖性的胞质成分向膜的募集在进化中是保守的。在微粒体结合试验中评估了pfGAPDH的两个重叠片段（第1-192位和第133-337位残基）。我们发现N端片段竞争性抑制Rab2刺激的pfGAPDH募集。因此，介导进化上保守的Rab2依赖性膜募集的结构域位于GAPDH的N端。这些结果共同表明，pfGAPDH在恶性疟原虫中发挥非糖酵解功能，可能包括在囊泡运输和顶端细胞器生物发生中的作用。
