Lysosomal biogenesis & function in African trypanosomes

超出提供的空间。非洲锥虫是一种寄生原生动物，会导致人类昏睡病。在这些古老的真核生物中，溶酶体的生物发生机制知之甚少，p67是一种重要的膜糖蛋白，主要位于溶酶体中，但也存在于鞭毛袋和内体中。它以一种阶段特异性的方式运输到溶酶体，在血液细胞中，它的N-糖链通过合成聚乳糖胺(PNAL)链来修饰。在锥体中，分泌和内吞在鞭毛袋中必然相交；作为一种存在于此交叉处的必需蛋白质，p67是研究这些关键过程如何塑造溶酶体的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靶向是由其细胞质区域中的特定信号介导的；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体分泌/内吞运输的新方面的理解，以及对数百万年真核进化过程中保守的蛋白质靶向机制的理解。在AIM#1中，膜蛋白报告基因将被融合到p67细胞质结构域，突变将被用来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运输机制一部分的相互作用蛋白。胞质结构域将以TAP-Tag融合的形式在锥体中表达，并将通过亲和层析分离原位复合体。细胞质结构域也将被用作酵母双杂交筛选中的遗传诱饵，以识别相互作用蛋白的基因。在目标3中，我们将使用RNAi沉默来检测p67在溶酶体功能中的作用。初步的RNAi实验表明，p67是血流锥体中的一种必需蛋白质，我们的假设是，p67提供了一种保护糖基化作用，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来产生缺乏合成不寻常的pNAL N-聚糖的细胞系，这种不寻常的pNAL N-聚糖是在血液寄生虫的鞭毛袋/内体系统的p67和其他膜蛋白上发现的。突变体将被选择为未能内吞荧光番茄凝集素，然后被鉴定为溶酶体/内吞过程中的一般缺陷，以及p67靶向和功能的特定缺陷。表演网站========================================Section End===========================================

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===========================================