GIARDIA LAMBLIA CYSTEINE PROTEASES: TRAFFICKING, LOCALIZATION, AND FUNCTION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 贾第虫是细胞生物学的重要模型，因为它代表了基于保守基因序列分析的最基本的真核谱系。 贾第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，和蛋白质运输室，如一个经典的高尔基体和分泌颗粒。 相反，贾第虫采用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种新的蛋白质区室，似乎充当ER和内体。 使用荧光素酶结构，共聚焦显微镜，和超微结构分析，我们已经表明，贾第虫半胱氨酸蛋白酶定位到一个tubulovesicular网络与ER样结构周边的核周膜。 标记的蛋白质被迅速内吞到该隔室中并被这些贾第虫半胱氨酸蛋白酶降解。 因此，似乎贾第鞭毛虫含有过渡内膜结构，可能早于内吞/溶酶体功能和内质网的区室化。 贾第虫半胱氨酸蛋白酶可以起到分解内吞营养物的作用。 然而，目前尚不清楚贾第虫半胱氨酸蛋白酶家族的哪些成员负责这种活性。 可以使用FPLC从贾第虫裂解物中分离和纯化半胱氨酸蛋白酶活性。 然后可以将该裂解物级分提交用于质谱分析以确定负责该新活性的基因产物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.