Fierro Manuel A, Muheljic Ajla, Sha Jihui, Wohlschlegel James A, Beck Josh R
bioRxiv. 2023 Jul 12:2023.07.12.548774. doi: 10.1101/2023.07.12.548774.
Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric ex port el ement (PEXEL)/Host Targeting Motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N-terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here we report that the non-exported parasitophorous vacuole protein UIS2 contains a PEXEL motif that is processed in the blood-stage. While the N-termini of exported proteins containing the PEXEL and immediately downstream ∼10 residues is sufficient to mediate translocation into the RBC, the equivalent UIS2 N-terminus does not promote export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RILτDE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N-terminus mediates export, not PEXEL processing . Prompted by this observation, we further show that PEXEL sequences in the N-termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export.
Host erythrocyte remodeling by malaria parasite exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite , most exported proteins undergo proteolytic maturation via recognition of the pentameric ex port el ement (PEXEL)/Host Targeting motif by the aspartic protease Plasmepsin V (PMV) which exposes a mature N-terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing by PMV, reinforcing that features of the mature N-terminus, and not PEXEL cleavage, identify cargo for export cargo. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.
专性细胞内疟原虫通过效应蛋白输出显著重塑其红细胞宿主，以创造一个生存小生境。大多数输出蛋白含有一个五聚体输出元件（PEXEL）/宿主靶向基序，该基序在寄生虫内质网中被天冬氨酸蛋白酶疟原虫天冬氨酸蛋白酶V（PMV）切割。这一加工事件暴露出转运到宿主细胞所需的成熟N端，且已知在非输出蛋白中不会发生。在此，我们报告非输出的寄生泡蛋白UIS2含有一个在血液阶段被加工的PEXEL基序。虽然含有PEXEL的输出蛋白的N端及紧邻下游约10个残基足以介导转运到红细胞中，但等效的UIS2 N端并不能促进报告基因的输出。奇怪的是，UIS2 PEXEL在第四个位置含有一个不寻常的天冬氨酸，该天冬氨酸构成PEXEL切割后最末端的N端残基（P1'，RILτDE）。使用一系列嵌合报告基因融合体，我们表明P1'位的天冬氨酸允许PMV加工，但废除输出。此外，将这个单一的UIS2残基突变为丙氨酸可实现输出，这进一步证明成熟的N端介导输出，而非PEXEL加工。受这一观察结果的启发，我们进一步表明其他非输出的棒状体蛋白N端的PEXEL序列也被加工，这表明PMV可能是一种比以前所认识到的更普遍的分泌成熟酶，类似于相关顶复门生物中的直系同源物。我们的发现为标记用于输出的蛋白的独特N端限制提供了新的见解。
疟原虫输出的效应蛋白对宿主红细胞的重塑对于寄生虫的生存和疾病发病机制至关重要。在最致命的疟原虫中，大多数输出蛋白通过天冬氨酸蛋白酶疟原虫天冬氨酸蛋白酶V（PMV）识别五聚体输出元件（PEXEL）/宿主靶向基序进行蛋白水解成熟，从而暴露出有利于输出到红细胞宿主细胞中的成熟N端。虽然PEXEL加工被认为是输出蛋白的独特标志，但我们证明PEXEL基序存在于非输出蛋白中并被加工。重要的是，我们表明PEXEL基序可变的第四个位置的特定残基尽管允许被PMV加工，但会抑制输出，这进一步证明成熟N端的特征而非PEXEL切割识别用于输出的货物。这为进一步探究PEXEL基序的性质和进化打开了大门。
