Independent Analysis of the Flagellum Surface and Matrix Proteomes Provides Insight into Flagellum Signaling in Mammalian-infectious Trypanosoma brucei

Abstract

The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for hostparasite signaling.

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

“…The TbNMT knockdown resulted in a shift of electrophoretic mobility of POMP39 and a concomitant change of localization, whereas the putative palmitoylation was experimentally addressed in a previous study (Emmer et al, 2011). In line with the global proteomics studies mentioned above Panigrahi et al, 2009;Oberholzer et al, 2011), we show that a fraction of the protein localizes to the flagellum, whereas another fraction is stably associated with the MOM.…”

“…60% identity) was in our proteomics study never found in the mitochondrial fraction and had the relative maximal intensity in the crude ER fraction (Niemann et al, 2013). Furthermore, it was not detected in the flagellar proteome (Oberholzer et al, 2011).…”

“…POMP39 was also identified in the flagellar matrix proteome of BSF trypanosomes (Oberholzer et al, 2011), hinting that it might be dually localized. POMP39 contains both a predicted myristoylation and palmitoylation site (99.97% and ≥95% certainty, respectively).…”

“…Besides POMP39 there is a single protein, POMP3 (Tb927.9.2670) that is predicted to be dual acylated by myristoylation and palmitoylation. POMP3 has two predicted transmembrane helices and has neither been found in the flagellar nor the mitochondrial proteome before Oberholzer et al, 2011;Panigrahi et al, 2009), but the palmitoylation was experimentally confirmed (Emmer et al, 2011). The function of POMP3 is unknown and the protein is only present in trypanosomatids.…”

“…As a consequence, African trypanosomes have recently been subjected to sub-cellular and suborganellar proteomics approaches scrutinizing the protein inventory of mitochondria and their membranes Niemann et al, 2013;Panigrahi et al, 2009), the mitochondrial ribosomes (Zikova et al, 2009) and the respiratory chain . Moreover, the composition of the flagellum has also been analyzed in detail (Broadhead et al, 2006;Oberholzer et al, 2011). These comprehensive proteomics approaches have greatly increased the knowledge about the sub-cellular localization of a large fraction of trypanosomal proteins and resulted in an increasing number of proteins that have been detected in two or more cellular compartments.…”