The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei

Abstract

Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P2 in T. brucei. We find a low steady state level of PI(3,5)P2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P2 pool leading to rapid cell death. TbFab1 and PI(3,5)P2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P2 metabolism, the PI(3,5)P2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology.

Late ESCRT Machinery Mediates The Recycling And Rescue of Invariant Surface Glycoprotein 65 inTrypanosoma brucei

“…Validated markers for each compartment are: EE, TbRab5A/B (Pal, Hall, Nesbeth, Field, & Field, 2002); RE, TbRab11 Umaer et al, 2018); LE, TbRab7 Silverman et al, 2011), TbRab28 (Lumb et al, 2011), TbVps4 (Silverman et al, 2013), TbVps23 Silverman et al, 2013), and TbVps24 (this work); Lyso, p67 (Alexander et al, 2002) and TbCatL (Koeller & Bangs, 2018;Peck et al, 2008). TbFab1 localizes to both LE and Lyso (Gilden et al, 2017).…”

“…Knock down of the PI(3)P-5 kinase, TbFab1, in BSF trypanosomes causes a reduction in PI(3,5)P 2 levels and a complete blockage of ISG65 turnover in the lysosome (Gilden et al, 2017). Mammalian Vps24 and related ESCRT III components have been shown to bind PI(3,5)P 2 and PI(P3,4)P 2 (Catimel et al, 2008;Whitley et al, 2003).…”

Phosphoinositide signaling and regulation in Trypanosoma brucei: Specialized functions in a protozoan pathogen

“…This set of synthesis, cleavage, and modifying enzymes (hereafter referred as PIP/IP-related proteins) produces at least 11 different PIP and IP metabolites ( Fig 1C), some of which have been detected in T. brucei via immunofluorescence or mass spectrometry methods [7,14,15] or predicted to exist based on in vitro enzymatic studies [6,13,18]. T. brucei PIP and IP kinases and phosphatases with different specificities are distributed in distinct subcellular locations, e.g., plasma membrane, endosomes, and nucleus [5,7,9,10,12] (Table 1). The subcellular distribution of PIPs, IPs, and related proteins in T. brucei indicates that they function as a regulatory system in addition to their role in the synthesis of membrane or glycoconjugate structures.…”

“…The subcellular distribution of PIPs, IPs, and related proteins in T. brucei indicates that they function as a regulatory system in addition to their role in the synthesis of membrane or glycoconjugate structures. This is evidenced by the numerous cellular processes that are affected by knockdown or mutation of genes encoding PIP/IP-related proteins [6,9,10,12,18,19] (Table 1). This regulatory system relies primarily on the activity of PIP and IP kinases and phosphatases, which control the phosphorylation and turnover of PIP and IP metabolites, and on the ability of these metabolites to interact with proteins and thus regulate protein function.…”

“…PIPs and IPs interact with proteins or RNA and regulate numerous cellular functions in eukaryotes. As detailed below, these metabolites and related enzymes function as a regulatory system with essential roles in T. brucei metabolism and development [6], trafficking and organelle biogenesis [9][10][11], Ca 2+ signaling [12], and immune evasion mechanisms [5,7].…”