The Trypanosome Exocyst:A Conserved Structure Revealing a New Role in Endocytosis

Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities

SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes

SUMOylation is a post¿translational modification that positively regulates monoallelic expression of the trypanosome variant surface glycoprotein (VSG). The presence of a highly SUMOylated focus associated with the nuclear body, where the VSG gene is transcribed, further suggests an important role of SUMOylation in regulating VSG expression. Here, we show that SNF2PH, a SUMOylated plant homeodomain (PH)¿transcription factor, is upregulated in the bloodstream form of the parasite and enriched at the active VSG telomere. SUMOylation promotes the recruitment of SNF2PH to the VSG promoter, where it is required to maintain RNA polymerase I and thus to regulate VSG transcript levels. Further, ectopic overexpression of SNF2PH in insect forms, but not of a mutant lacking the PH domain, induces the expression of bloodstream stage¿specific surface proteins. These data suggest that SNF2PH SUMOylation positively regulates VSG monoallelic transcription, while the PH domain is required for the expression of bloodstream¿specific surface proteins. Thus, SNF2PH functions as a positive activator, linking expression of infective form surface proteins and VSG regulation, thereby acting as a major regulator of pathogenicity.The authors thank Dr. Alicia Barroso Del Jesus for excellent assistance and input with NSG methodology at the Genomic Unit and Dr. Laura Montosa at the Microscopy Unit (IPBLN-CSIC). This work was supported by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (RTI2018-098834-B-I00) and the Wellcome Trust (WTI 204697/Z/16/Z to MCF) and thegrant from the Argentinian National Agency for Promotion of Scientific and Technological Research to VEA (PICT/2016/0465)

Proteomics uncovers novel components of an interactive protein network supporting RNA export in trypanosomes

In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation

Life and times:synthesis, trafficking, and evolution of VSG

Evasion of the acquired immune response in African trypanosomes is principally mediated by antigenic variation, the sequential expression of distinct variant surface glycoproteins (VSGs) at extremely high density on the cell surface. Sequence diversity between VSGs facilitates escape of a subpopulation of trypanosomes from antibody-mediated killing. Significant advances have increased understanding of the mechanisms underpinning synthesis and maintenance of the VSG coat. In this review, we discuss the biosynthesis, trafficking, and turnover of VSG, emphasising those unusual mechanisms that act to maintain coat integrity and to protect against immunological attack. We also highlight new findings that suggest the presence of unique or highly divergent proteins that may offer therapeutic opportunities, as well as considering aspects of VSG biology that remain to be fully explored

Quantitative proteomics by SILAC identifies altered surface protein expression mediated by exocyst knockdown.

<p>(A) Volcano plot of protein abundance changes at 36h post Sec15 RNAi induction. -log₁₀ transformed SILAC ratios are plotted against -log₁₀ transformed standard deviation. Data points representing protein groups significantly altered after 36 hours are labeled; ISG65 and ISG75 paralogs in dark green, other potential surface proteins in light green, mitochondrial proteins in red, cytoplasmic proteins in blue, lysosomal proteins in yellow, others in grey. AQP1: Aquaporin1, TryS: trypanothione synthetase; PTP1 interactor: Protein-tyrosine-phosphatase1-interacting protein; PAD1: Protein associated with differentiation 1; CoA transferase: Succinyl-coA:3-ketoacid-coenzyme A transferase; GCVL-2: Dihydrolipoyl dehydrogenase. (B) Predicted cellular localisation of proteins upregulated in Sec15 RNAi cells 36h post induction as reported by GO terms or as predicted by TMHMM2 for <i>trans-</i>membrane domains and PredGPI for GPI-anchor addition C-terminal signal sequences [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006063#ppat.1006063.ref089" target="_blank">89</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006063#ppat.1006063.ref090" target="_blank">90</a>]. Numbers of proteins in each category are in parenthesis.</p

The exocyst is required for endocytosis.

<p>(A) Uninduced and induced Sec15 and Exo99::RNAi cells were allowed to accumulate FITC-conjugated ConA at the indicated temperatures. Fixed cells counterstained with DAPI (blue) and anti-p67 antibody for the lysosome (green). Both Sec15 and Exo99::RNAi lines show a defect in delivery of ConA to the lysosome: at 12°C and 37°C ConA remains predominantly localised at the flagellar pocket. (B) Dynamics of ConA uptake at 37°C 48h post RNAi induction. In control cells, ConA accumulates in a p67-positive compartment within 30 min. By contrast Sec15 and Exo99::RNAi cells exhibit a marked delay in delivery of ConA to the lysosome. In all images DAPI was used to visualise DNA (blue). Scale bar, 5 μm. (C) Quantitation of ConA lysosomal delivery at 37°C 48h post induction. N = 25 cells per time point per duplicate experiment. (D) Representative electron micrograph of curved flagellar pocket membrane coated by clathrin. Arrowheads point at clathrin coat.</p

The exocyst functions in endocytosis in human cells.

<p>(A) Immunoblots of lysates prepared from HeLa cells treated with either scrambled or EXOC6 siRNA smart pools as shown in panel B, probed with anti-EXOC6 or anti-GAPDH as indicated. Knockdown levels of EXOC6 were quantified from 3 experiments of this type and are presented as a ratio of EXOC6/GAPDH signals. (B) HeLa cells depicting transferrin uptake. Cells treated with either scrambled or EXOC6 siRNA were imaged after the uptake of labelled transferrin (10 min). Representative fields of cells from 3 independent replicates. (C) The fluorescence intensity of >100 cells for each condition was determined using ImageJ and the data compared using an unpaired t test. *** P value < 0.0001. (D) The image intensity of >100 cells of each condition were binned into two groups, those with a signal intensity >500 arbitrary units, and those <500. Knockdown of EXOC6 was found to significantly increase the fraction of cells with a signal intensity <500 (** P value <0.01), consistent with a decrease in transferrin endocytosis.</p

The exocyst is not required for endomembrane system maintenance or morphology.

<p>Immunofluorescence microscopy analysis of selected organelle markers or trafficking pathways for Sec15::RNAi and Exo99::RNAi cells at 24 (D1) and 48h (D2) post induction. Cells were stained with specific antibodies against GRASP (red), p67 (green), clathrin heavy chain (red) or Rab11 (red) and counter-stained with DAPI to visualise DNA (blue). The morphology and protein expression levels of all molecular markers remained stable. Scale bar, 5 μm.</p

Identification of nine exocyst subunits in trypanosomes.

<p>(A) Immunoisolation from PCF cells constitutively expressing Sec15::GFP or Exo99::GFP. Pullouts were performed with the following buffers; (1) 20mM HEPES pH 7.4, 500mM NaCl 5% Triton and protease inhibitors; (2) 20mM HEPES pH 7.4, 250mM NaCitrate 5% CHAPS and protease inhibitors. (B) Immunofluorescence microscopy of PCF cells expressing Sec15::GFP and Exo99::GFP (green), BSF cells expressing Sec15::HA and Exo99::HA (red) and PCF cells co-expressing Sec15::GFP and Exo99::HA. DAPI was used to visualise DNA (blue). Scale bar, 5 μm. In both lifecycle stages Sec15 and Exo99 localise to the region between the nucleus and kinetoplast where the organelles of the endocytic and secretory pathways are found. (C) Immunofluorescence of PCF cells expressing Sec5::GFP and Exo84::GFP (green). Sec5 and Exo99 localise to the same region which indicates that they are part of the same complex. (D) Predicted secondary structure of Exo99 according to PSIPRED. The horizontal black line represents the polypeptide span with the N-terminus to the left, the y-axis indicates the confidence score of predicted secondary structure. Predicted α-helix are shown in red, predicted β-sheet in blue.</p

The exocyst is required for normal membrane trafficking.

<p>Representative transmission electron micrographs showing the effect of exocist subunit ablation 48h post-RNAi induction in BSF cells. The steady-state flagellar pocket has a small overall volume (A); ablation of Exo99 and Sec15 causes pocket enlargement (B, E), over-production of large VSG-coated vesicles inside the flagellar pocket (C, F, G), failure in cytokinesis (as illustrated by multiple nuclei in panels D and I) and ER hypertrophy (H). Despite large flagellar pocket volumes, indicative of endocytosis defect, clathrin is recruited to the surface membrane and able to assemble into coated pits and lattices (B, F).</p