Global Analysis of Protein Palmitoylation in African Trypanosomes

Abstract

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to longchain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.

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

“…The cysteine residue on position three (C3) is a predicted palmitoylation site with ≥95% confidence level when analyzed using the CSSPalm algorithm (Ren et al, 2008). Consistent with this prediction, POMP39 was identified as one of 124 proteins enriched by streptavidin chromatography following an acyl-biotin exchange-reaction of whole cell lysates (Emmer et al, 2011). Notably, this streptavidin-capture of POMP39 was hydroxylaminesensitive.…”

Emerging roles for protein S-palmitoylation in Toxoplasma biology

“…More than 400 putative P. falciparum palmitoylated proteins were identified in the highly enriched fraction (Jones et al, 2012). This is significantly more than what has been reported to date for the palmitome of Saccharomyces cerevisiae (50 proteins) (Roth et al, 2006), T. brucei (124 proteins) (Emmer et al, 2011) and mammalian cells (between 125 and 260 proteins) (Kang et al, 2008;Martin and Cravatt, 2009;Yount et al, 2010). These findings led to the assumption that P. falciparum makes substantial use of palmitoylation, wider than previously thought, with implications for many diverse biological processes including signalling, development, invasion as well as cytoadherence and drug resistance (Jones et al, 2012).…”

“…Despite the existence of eight Golgi-localised DHHCs, two of them probably exhibit unique features, as the corresponding genes could not be disrupted (Frenal et al, 2013). This is the first organism for which five PATs appear to individually fulfil some critical functions whereas all of the yeast or T. brucei PATs are apparently dispensable or functionally redundant (Roth et al, 2006;Emmer et al, 2011). DHHC7 is the PAT that localises to the rhoptries of T. gondii, P. falciparum and P. berghei (Frenal et al, 2013).…”

“…In the procyclic form of the protozoan parasite Trypanosoma brucei, an experimental palmitome analysis was performed by an Acyl-Biotin Exchange (ABE) method (Emmer et al, 2011) ( Fig. 2A).…”

“…For example, the repertoire of DHHCs in S. cerevisiae (Mitchell et al, 2006) includes seven enzymes, whereas T. brucei possesses 12 (Emmer et al, 2011) and Arabidopsis thaliana has 23 (Batistic, 2012), the same number as found in humans (Putilina et al, 1999). The P. falciparum genome encodes 12 DHHC-containing proteins, whereas T. gondii possesses the largest family in the Apicomplexa with 18 genes, 16 of which are expressed in the tachyzoite stage (Frenal et al, 2013).…”