Lysosomal biogenesis & function in African trypanosomes

非洲锥虫是一种寄生原生动物，会导致人类昏睡病。鲜为人知 在这些古老的真核生物中，p67是一种重要的膜糖蛋白。 主要在溶酶体中，但也在鞭毛袋和内体中。它以一种形式贩卖到溶酶体 在血液细胞中，其N-糖链通过合成聚乳糖胺进行修饰 (PNAL)链。在锥虫体内，分泌和内吞作用在鞭毛袋中必然相交；作为 存在于这个交叉点的必需蛋白质p67是研究这些关键过程如何 溶酶体的形态、生物发生和功能。我们将研究p67靶向的信号和机制。 它在溶酶体结构/功能中的作用。我们的基本假设是：i)p67靶向是由 以及ii)p67对于溶酶体的生物发生和功能是必不可少的。 这项工作将扩大我们对锥虫分泌/内吞运输的新方面的理解， 以及在数百万年的真核生物进化过程中保存下来的蛋白质靶向机制。 在AIM#1中，膜蛋白报告基因将与p67细胞质结构域融合，并发生突变 用于鉴定介导溶酶体靶向的特定序列基序。在AIM#2中，细胞质 结构域将被用作诱饵，以确定作为p67贩运机制一部分的相互作用蛋白。这个 胞质结构域将以TAP-Tag融合的形式在锥体中表达，原位复合体将被 用亲和层析法分离。胞质结构域也将在酵母中用作遗传诱饵。 双杂交筛选用于识别相互作用蛋白的基因。在目标3中，我们将使用RNAi沉默来检查 P67在溶酶体功能中的作用。初步的RNAi实验表明，p67是一种必需的蛋白质。 我们的假设是，p67提供了一种保护性的糖基化，以防止内部 水解酶和潜在的裂解宿主血清因子。在目标4中，我们将使用化学诱变 为了产生在p67和其他基因上发现的不寻常的pNAL N-糖链合成不足的细胞系 血流寄生虫鞭毛囊/内体系统的膜蛋白。变种人将会是 由于未能内吞荧光番茄凝集素而被选中，然后被鉴定为在 溶酶体/内吞过程，以及p67靶向和功能的特定缺陷。

African trypanosomes are parasitic protozoa that cause sleeping sickness in humans. Little is known about lysosomal biogenesis in these ancient eukaryotes, p67 is an essential membrane glycoprotein located primarily in the lysosome, but also in the flagellar pocket and endosomes. It trafficks to the lysosome in a stage-specific manner, and in bloodstream cells its N-glycans are modified by synthesis of poly-lactosamine (pNAL) chains. In trypanosomes secretion and endocytosis obligately intersect in the flagellar pocket; as an essential protein that exists at this intersection p67 is a unique tool for studying how these critical processes shape lysosomal biogenesis and function. We will study of the signals and machinery of p67 targeting and its role in lysosomal structure/function. Our underlying hypotheses are: i) that p67 targeting is mediated by specific signals in its cytoplasmic domain; and ii) that p67 is essential for lysosomal biogenesis and function. This work will expand our understanding of the novel aspects of trypanosomal secretory/endocytic trafficking, and of mechanisms of protein targeting conserved over many millions of years of eukaryotic evolution. In AIM #1 membrane protein reporters will be fused to the p67 cytoplasmic domain and mutagenesis will be used to identify the specific sequence motifs that mediate lysosomal targeting. In AIM #2 the cytoplasmic domain will be used as bait to identify interactive proteins that are part of the p67 trafficking machinery. The cytoplasmic domain will be expressed in trypanosomes as a TAP-tag fusion and in situ complexes will be isolated by affinity chromatography. The cytoplasmic domain will also be used as genetic bait in a yeast two-hybrid screen to identify genes for interactive proteins. In Aim #3 we will use RNAi silencing to examine the role of p67 in lysosomal function. Preliminary RNAi experiments indicate that p67 is an essential protein in bloodstream trypanosomes and our hypothesis is that p67 provides a protective glycocalyx against internal hydrolases and against potentially lytic host serum factors. In Aim #4 we will employ chemical mutagenesis to generate cell lines deficient in synthesis of the unusual pNAL N-glycans that are found on p67 and other membrane proteins of the flagellar pocket/endosomal system in bloodstream parasites. Mutants will be selected for failure to endocytose fluorescent tomato lectin and then characterized for general deficiencies in lysosomal/endocytic processes, and for specific defects in p67 targeting and function.