GIARDIA LAMBLIA CYSTEINE PROTEASES: TRAFFICKING, LOCALIZATION, AND FUNCTION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 贾第虫是一个重要的细胞生物学模型，因为它代表了基于保守基因序列分析的最基本的真核生物谱系。贾第鞭毛虫滋养体缺乏许多典型的真核细胞器，如线粒体、过氧化物酶体和蛋白质运输间隔，如经典的高尔基体和分泌颗粒。取而代之的是，贾第虫使用一个简单的内膜系统将蛋白质输出到不同的细胞内位置。我们在贾第鞭毛虫中发现了一个新的蛋白隔室，它似乎是内质网和内体。 利用报告构建、共聚焦显微镜和超微结构分析，我们已经证明了贾第鞭毛虫半胱氨酸蛋白酶定位于一个位于核周膜周围的具有ER样结构的管状囊泡状网络中。标记的蛋白质迅速内吞到这个隔室，并被这些贾第鞭毛虫半胱氨酸蛋白酶降解。因此，贾第鞭毛虫含有一种过渡性的膜内结构，这种结构可能早于内吞/溶酶体功能和内质网的划分。贾第鞭毛虫半胱氨酸蛋白酶可能起到分解内吞营养物质的作用。然而，目前还不清楚贾第鞭毛虫半胱氨酸蛋白酶家族的哪些成员负责这一活动。用FPLC可以从贾第鞭毛虫裂解物中分离纯化半胱氨酸蛋白酶活性。然后，这些裂解产物可以提交给质谱分析，以确定负责这一新活性的基因产物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Giardia is an important model for cell biology because it represents the most basal of eukaryotic lineages based on sequence analysis of conserved genes. Giardia trophozoites lack many typical eukaryotic organelles such as mitochondria, peroxisomes, and protein trafficking compartments such as a classical Golgi apparatus and secretory granules. Instead, Giardia employs a simple endomembrane system to export proteins to distinct intracellular locations. We have discovered a novel protein compartment in Giardia that appears to serve as ER and endosome. Using reporter-constructs, confocal microscopy, and ultrastructural analysis, we have shown that Giardia cysteine proteases localize to a tubulovesicular network with an ER-like structure peripheral to the perinuclear membrane. Labeled proteins are rapidly endocytosed into this compartment and degraded by these Giardia cysteine proteases. It therefore appears that Giardia contains a transitional endomembranous structure that may pre-date compartmentalization of endocytic/lysosomal functions and the endoplasmic reticulum. Giardia cysteine proteases may function to break down endocytosed nutrients. However, it remains unclear which members of the Giardia cysteine protease family are responsible for this activity. Cysteine protease activity can be isolated and purified from Giardia lysate using FPLC. This lysate fraction can then be submitted for mass spectrometry analysis to determine the gene products responsible for this novel activity.