Characterisation of the plasma membrane subproteome of bloodstream form Trypanosoma brucei

Abstract

Proteome analysis by conventional approaches is biased against hydrophobic membrane proteins, many of which are also of low abundance. We have isolated plasma membrane sheets from bloodstream forms of Trypanosoma brucei by subcellular fractionation, and then applied a battery of complementary protein separation and identification techniques to identify a large number of proteins in this fraction. The results of these analyses have been combined to generate a subproteome for the pellicular plasma membrane of bloodstream forms of T. brucei as well as a separate subproteome for the pellicular cytoskeleton. In parallel, we have used in silico approaches to predict the relative abundance of proteins potentially expressed by bloodstream form trypanosomes, and to identify likely polytopic membrane proteins, providing quality control for the experimentally defined plasma membrane subproteome. We show that the application of multiple high-resolution proteomic techniques to an enriched organelle fraction is a valuable approach for the characterisation of relatively intractable membrane proteomes. We present here the most complete analysis of a protozoan plasma membrane proteome to date and show the presence of a large number of integral membrane proteins, including 11 nucleoside/nucleobase transporters, 15 ion pumps and channels and a large number of adenylate cyclases hitherto listed as putative proteins.

Host-Parasite Interactions in Trypanosomiasis: on the Way to an Antidisease Strategy

“…Several genome projects are also under way for T. brucei gambiense, T. congolense, and T. vivax (62). Besides the significant success of the T. cruzi proteome project (6), the recent description of the T. brucei plasma membrane subproteome was a unique breakthrough (24,60). These achievements should bring insights into host-parasite interactions and the identification of new vaccine candidates or chemotherapeutic targets.…”

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

“…POMP39 has neither a predicted transmembrane helix nor a signal anchor. The polypeptide is strongly overexpressed in the BSF, as evident from proteomics studies (Bridges et al, 2008;Gunasekera et al, 2012).…”

Fluorescence two-dimensional difference gel electrophoresis for biomaterial applications

“…Another modification, known as Blue Native-DiGE, has been employed to improve the resolution of hydrophobic proteins, and also gives some indication of the differences in native protein structures (discussed in Dani & Dencher 2008). Similarly, it has recently been possible to adapt a technique for improved two-dimensional electrophoretic display of hydrophobic proteins (described in Bridges et al 2008) for minimal labelling DiGE (D. J. Bridges & R. J. S. Burchmore 2006, unpublished data). Such continual developments should enable flexibility for novel applications of the technique to the biomaterials field, such as the investigation of cell stress in response to materials with redox DiGE, and global analysis of the membrane and surface markers expressed over time by differentiating cells on structured substrata.…”