Membrane interactions of GPI-PLC in vitro and in Trypanosoma brucei
Hardin, Clyde Franklin
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Trypanosoma brucei is the causative agent of human African sleeping sickness. T. brucei is covered with a surface coat that primarily consists of variant surface glycoprotein (VSG). A glycosyl phosphatidylinositol (GPI) anchors the VSG protein to the extracellular surface of the plasma membrane. T. brucei contains a GPI-specific phospholipase C (GPI-PLCp) that is capable of cleaving GPI-anchors and GPI-intermediates. GPI-PLCp is an integral membrane protein however, how the protein binds to membranes, and where it localizes in the cell, is not known. The purpose of this study is to identify regions of GPI-PLCp that bind to membranes and to identify where the enzyme localizes in vivo. The enzyme does not contain any transmembrane domains or sequences homologous to characterized membrane-binding domains. Therefore, in an effort to identify the membrane-binding regions of GPI-PLCp, amino and carboxyl-terminal truncations of the enzyme were made. Constructs were assayed for their ability to target a soluble reporter protein, green fluorescent protein (GFP), to T. brucei microsomes in vitro. Amino acids 60-120 of GPI-PLCp targeted GFP to microsomes. In vivo experiments indicate that GPI-PLCp colocalizes with the glycosomal protein, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To further study where the enzyme localizes in vivo, T. brucei glycosomes were isolated by 15-60 % sucrose-gradient sedimentation of microbodies isolated by differential centrifugation. Fractions that contained GPI-PLCp activity, also contained the glycosomal protein, aldolase. These results indicate that GPI-PLCp localizes to the glycosome in T. brucei. We propose that amino acids 60-120 contribute to the association of GPI-PLCp with glycosomes.