Dataset Information

Trypanosoma brucei SURFEOME: a validated surface proteome of the host-parasite interface

ABSTRACT: Surface membrane organization and composition is key to cellular function, and membrane proteins serve many essential roles in endocytosis, secretion and cell recognition. The surface of parasitic organisms, however, is a double-edged sword; this is the primary interface between parasites and their hosts, and those crucial cellular processes must be carried out while avoiding elimination by the host immune defenses. For extracellular African trypanosomes, the surface is partitioned such that all endo- and exocytosis is directed through a specific membrane region, the flagellar pocket, in which it is thought the majority of invariant surface proteins reside. However, very few of these proteins have been identified, severely limiting functional studies, and hampering the development of potential treatments. Here we used an integrated biochemical, proteomic and bioinformatic strategy to identify surface components of the human parasite Trypanosoma brucei. This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival. Molecules with receptor-like properties are almost exclusively parasite-specific, whereas transporter-like proteins are conserved in model organisms. Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins, and as expected, the majority present at the flagellar pocket. Moreover, the largest systematic analysis of trypanosome surface molecules to date provides evidence that the cell surface is compartmentalized into three distinct domains with free diffusion of molecules in each, but selective, asymmetric traffic between. This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Trypanosoma Brucei

TISSUE(S): Blood Cell, Cell Culture

DISEASE(S): Trypanosomiasis

SUBMITTER: Catarina Gadelha

LAB HEAD: Catarina Gadelha

PROVIDER: PXD002180 | Pride | 2015-06-15

REPOSITORIES: Pride

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Publications

Architecture of a Host-Parasite Interface: Complex Targeting Mechanisms Revealed Through Proteomics.

Gadelha Catarina C Zhang Wenzhu W Chamberlain James W JW Chait Brian T BT Wickstead Bill B Field Mark C MC

Molecular & cellular proteomics : MCP 20150430 7

Surface membrane organization and composition is key to cellular function, and membrane proteins serve many essential roles in endocytosis, secretion, and cell recognition. The surface of parasitic organisms, however, is a double-edged sword; this is the primary interface between parasites and their hosts, and those crucial cellular processes must be carried out while avoiding elimination by the host immune defenses. For extracellular African trypanosomes, the surface is partitioned such that al ...[more]

PMID: 25931509

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Project description:Variant Surface Glycoproteins (VSG) coat parasitic African trypanosomes and underpin antigenic variation and immune evasion. This VSG is a super-abundant virulence factor that is subject to post-transcriptional gene expression controls mediated via the VSG 3’-untranslated region (3’-UTR). To identify positive VSG regulators in bloodstream form cells, we used prior genome-scale screening data to prioritise mRNA binding protein (mRBPs) knockdowns that phenocopy VSG mRNA knockdown, displaying both loss-of-fitness and pre-cytokinesis accumulation. The top three candidate VSG regulators were CFB2 (cyclin F-box protein 2, Tb927.1.4650), MKT1 (Tb927.6.4770) and PBP1 (polyadenylate binding-protein binding-protein, Tb927.8.4540). Notably, CFB2 was recently found to regulate VSG transcript stability, and all three proteins were found to associate with each other. We used data-independent acquisition for accurate label-free quantification and deep proteome coverage to quantify expression profiles following depletion of each mRBP. Only CFB2 knockdown significantly reduced VSG expression and the expression of a reporter under the control of a VSG 3’-untranslated region (3’-UTR). CFB2 knockdown also triggered depletion of cytoplasmic ribosomal proteins, consistent with translation arrest observed when VSG synthesis is blocked. In contrast, PBP1 knockdown triggered depletion of CFB2, MKT1, and other components of the PBP1-complex. Finally, all three knockdowns triggered depletion of cytokinesis initiation factors, consistent with the cytokinesis defect that was confirmed here for all three knockdowns. Thus, genome-scale knockdown datasets facilitate the triage and prioritisation of candidate regulators. Quantitative proteomic analysis confirms 3’-UTR dependent positive control of VSG expression by CFB2 and interactions with additional mRBPs. Our results also reveal connections between VSG expression control by CFB2, ribosomal protein expression, and cytokinesis.

Project description:Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to mediate Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to mediate cellular processes such as mitosis and intracellular transport. In trypanosomatids, the kinesin superfamily has undergone a prominent expansion, resulting in one of the most diverse kinesin repertoires that includes the two kinetoplastid-restricted families X1 and X2. Here, we characterize in Trypanosoma brucei TbKifX2A, an orphaned X2 kinesin. TbKifX2A tightly interacts with TbPH1, a kinesin-like protein with a likely inactive motor domain, a rarely reported occurrence. Both TbKifX2A and TbPH1 localize to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that wraps around the flagellar pocket as it extends to the cell body anterior. The proximal proteome of TbPH1 revealed two other interacting proteins, the flagellar pocket protein FP45 and intriguingly another X2 kinesin, TbKifX2C. Simultaneous ablation of TbKifX2A/TbPH1 results in the depletion of FP45 and TbKifX2C and also an expansion of the flagellar pocket, among other morphological defects. TbKifX2A is the first motor protein to be localized to the MtQ. The observation that TbKifX2C also associates with the MtQ suggests that the X2 kinesin family may have co-evolved with the MtQ, both kinetoplastid-specific traits.cellular processes such as mitosis and intracellular transport. In trypanosomatids, the kinesin superfamily has undergone a prominent expansion, resulting in one of the most diverse kinesin repertoires that includes the two kinetoplastid-restricted families X1 and X2. Here, we characterize in Trypanosoma brucei TbKifX2A, an orphaned X2 kinesin. TbKifX2A tightly interacts with TbPH1, a kinesin-like protein with a likely inactive motor domain, a rarely reported occurrence. Both TbKifX2A and TbPH1 localize to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that wraps around the flagellar pocket as it extends to the cell body anterior. The proximal proteome of TbPH1 revealed two other interacting proteins, the flagellar pocket protein FP45 and intriguingly another X2 kinesin, TbKifX2C. Simultaneous ablation of TbKifX2A/TbPH1 results in the depletion of FP45 and TbKifX2C and also an expansion of the flagellar pocket, among other morphological defects. TbKifX2A is the first motor protein to be localized to the MtQ. The observation that TbKifX2C also associates with the MtQ suggests that the X2 kinesin family may have co-evolved with the MtQ, both kinetoplastid-specific traits.

			Action	DRS
	OMSSA_interact.pep.xml	Pepxml
	OMSSA_interact.prot.xml	Xml
	TbSurfeome-Dead1-F01.raw	Raw
	TbSurfeome-Dead1-F02.raw	Raw
	TbSurfeome-Dead1-F03.raw	Raw