Lysosomal biogenesis & function in African trypanosomes

超出所提供的空间。 非洲锥虫是一种寄生原生动物，会导致人类昏睡病。关于这些古老真核生物中的溶酶体生物发生知之甚少，p67 是一种必需的膜糖蛋白，主要位于溶酶体中，但也在鞭毛袋和内体中。它以特定阶段的方式运输至溶酶体，并且在血流细胞中，其 N-聚糖通过聚乳糖胺 (pNAL) 链的合成进行修饰。在锥虫中，分泌和内吞作用在鞭毛袋中必然相交；作为存在于该交叉点的必需蛋白质，p67 是研究这些关键过程如何塑造溶酶体生物发生和功能的独特工具。我们将研究 p67 靶向的信号和机制及其在溶酶体结构/功能中的作用。我们的基本假设是： i) p67 靶向是由其细胞质结构域中的特定信号介导的； ii) p67 对于溶酶体生物合成和功能至关重要。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，以及对数百万年真核进化中保守的蛋白质靶向机制的理解。 在 AIM #1 中，膜蛋白报告基因将与 p67 胞质结构域融合，并使用诱变来识别介导溶酶体靶向的特定序列基序。在 AIM #2 中，细胞质结构域将用作诱饵来识别作为 p67 运输机制一部分的相互作用蛋白。细胞质结构域将在锥虫中表达为 TAP-标签融合体，并通过亲和层析分离原位复合物。细胞质结构域还将用作酵母双杂交筛选中的遗传诱饵，以鉴定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 RNAi 实验表明 p67 是血流锥虫中的必需蛋白，我们的假设是 p67 提供了针对内部水解酶和潜在裂解性宿主血清因子的保护性糖萼。在目标#4中，我们将采用化学诱变来产生在血流寄生虫中的鞭毛袋/内体系统的p67和其他膜蛋白上发现的不寻常的pNAL N-聚糖合成方面存在缺陷的细胞系。将选择不能内吞荧光番茄凝集素的突变体，然后对溶酶体/内吞过程的一般缺陷以及 p67 靶向和功能的特定缺陷进行表征。表演网站==========================================章节结束==============================================

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================