Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A proximity labelling derived luminal proteome of the Trypanosoma brucei endoplasmic reticulum, Golgi apparatus and nuclear membrane

ABSTRACT: We employed proximity labelling to derive a proteome of the luminal content of the Trypanosoma brucei endoplasmic reticulum (ER) and Golgi apparatus. We exploited the abundant ER chaperone BiP (Binding-immunoglobulin protein) as ER BioID bait and compare to a truncated version of BiP (termed BiPN) devoid of ER retention and thus trafficked to the Golgi apparatus, as well as parental controls. Our approach covers the two major life cycle stages of T. brucei, i.e., the procyclic form (PCF) and the bloodstream form (BSF). Additionally, we probed the inner nuclear membrane which can be viewed as a specialised ER domain separated by the nuclear pore, via the membrane integral nucleoporin NUP65 as BioID bait in PCF. Altogether, our combined approaches devise an inventory of the T. brucei secretory pathway that complements existing subcellular protein maps derived by proteomics and imaging.

INSTRUMENT(S):

ORGANISM(S): Trypanosoma Brucei

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Trypanosomiasis

SUBMITTER: Martin Zoltner

LAB HEAD: Martin Zoltner

PROVIDER: PXD075517 | Pride | 2026-04-05

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	BIP_BSF_1.raw	Raw
	BIP_BSF_2.raw	Raw
	BIP_BSF_3.raw	Raw
	BIP_PCF_2.raw	Raw
	BIP_PCF_3.raw	Raw

Items per page:

1 - 5 of 24

Similar Datasets

Probing the trypanosome nuclear pore by TurboID proximity labelling – Part3

Project description:Previous BioID experiments targeting the nucleoporin (NUP) NUP158 (PMID: 36410438; PXD031245), as well as NUP110, NUP76, NUP96 (PMID: 39206942; PXD047268)and NUP75, transport factors MEX67 and Ran(PXD055934), indicated that proximity labelling delivers highly specific interactome data in the confined localisation of the nuclear pore, with a labelling radius well below the size of the nuclear pore. The resulting proximity data allowed to assign NUPs and nuclear transport factors to specific subregions of the pore. Here we extended this approach to target NUP98.

2025-05-07 | PXD059554 | Pride

Probing the trypanosome nuclear pore by TurboID proximity labelling – Part2

Project description:Previous BioID experiments targeting the nucleoporin (NUP) NUP158 (PMID: 36410438; PXD031245), as well as NUP110, NUP76 and NUP96 (PMID: 39206942; PXD047268) indicated that proximity labelling delivers highly specific interactome data in the confined localisation of the nuclear pore, with a labelling radius well below the size of the nuclear pore. The resulting proximity data allowed to assign NUPs and nuclear transport factors to specific subregions of the pore. Here we extended this approach to target NUP75 and transport factors MEX67 and Ran.

2024-09-20 | PXD055934 | Pride

Proximity labelling and cryomill affinity purification compared for Trypanosoma brucei proteins with unconfined, semi-confined and confined localisation

Project description:Identification of protein-protein interactions is a major contributor for understanding protein function and elucidating molecular and cellular mechanisms. Two strategies are currently popular for identification of protein interaction partners directly from the cellular environment. Affinity purification (pulldown) isolates the protein of interest with the aid of a matrix that specifically captures the target and potential partners. In BioID, the protein of interest is fused to biotin ligase facilitating proximity biotinylation and biotinylated proteins are isolated under stringent conditions with streptavidin. Whilst clear that these two methods can provide insight, it is also apparent that they can reveal distinct interactomes, but the basis for these differences is less obvious. We compare these methods using four different trypanosome proteins as baits: the cytoplasmic poly(A) binding proteins PABP1 and PABP2, mRNA export receptor MEX67, that shuttles between the nucleus and the cytoplasm with predominant localisation at the nuclear pores, and the nucleoporin NUP158.

2022-11-30 | PXD031245 | Pride

A unique mRNA decapping complex in trypanosomes

Project description:Removal of mRNA 5’ caps primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme DCP2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5´-3´exoribonuclease Xrn1. Kinetoplastida lack DCP2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. The enzyme is composed of a catalytic domain flanked by C-terminal and N-terminal extensions. Here we analyse the ALPH1 interactome by BioID proximity labelling for the full length protein and truncated versions in order to assign domain specific interactions. We show that Trypanosoma brucei ALPH1 acts in a complex composed of the trypanosome XRN1 ortholog XRNA and four proteins that are unique to Kinetoplastida.The interactome is validated by reverse experiments targeting T. brucei and T. cruzi XRNA by affinity capture and, additionally, the ALPH1 interacting CMGC-family kinase by BioID.

2023-06-14 | PXD038550 | Pride

Proteins associated wth Trypanosoma brucei HNRNPF/H

Project description:We aimed to find proteins associated with HNRNPH in bloodstream-form _Trypanosoma brucei_. We integrated a sequence encoding a cleavable TAP (Tandem Affinity Purification) tag into the genome upstream of, and in frame with, one _HNRNPH_ gene. The other allele was deleted. TAP-HNRNPH was purifed from triplicate lysates as shown under "Sample processing protocol". Sample processing protocol (30-5000chracters) The TAP-HNRNPH was bound to IgG beads, then released with Tobacco Etch Virus (TEV) protease. TAP-CFP served as the negative control. The His-tagged TEV protease was then removed using Ni-NTA-magnetic beads. Eluted proteins were separated on a 1.5 mm NuPAGE™ Novex™ 4-12 % Bis-Tris protein gel. For details see doi: 10.1371/journal.pone.0258903

2022-10-15 | PXD037289 | Pride

Essential PBP1-associated proteins of Trypanosoma brucei

Project description:Control of gene expression in kinetoplastids depends heavily on RNA-binding proteins that influence mRNA decay and translation. We previously showed that MKT1 interacts with PBP1, which in turn recruits LSM12 and poly(A) binding protein. MKT1 is recruited to mRNA by sequence-specific RNA-binding proteins, resulting in stabilisation of mRNA. We here show that PBP1, LSM12 and an additional 117-residue protein, XAC1 (Tb927.7.2780), are present in complexes that contain either MKT1 or MKT1L (Tb927.10.1490). All five proteins are present predominantly in the complexes, and there was evidence for a minor subset of complexes that contained both MKT1 and MKT1L. MKT1 appeared to be associated with many mRNAs, with the exception of those encoding ribosomal proteins. XAC1-containing complexes reproducibly contained RNAbinding proteins that were previously found associated with MKT1. In addition, however, XAC1- or MKT1- containing complexes specifically recruit one of the six translation initiation complexes, EIF4E6-EIF4G5; and yeast 2-hybrid assay results indicated that MKT1 interacts with EIF4G5. The C-terminus of MKT1L resembles MKT1: it contains MKT1 domains and a PIN domain that is probably not active as an endonuclease. MKT1L, however, also has an N-terminal extension with regions of low-complexity. Although MKT1L depletion inhibited cell proliferation, we found no evidence for specific interactions with RNA-binding proteins or mRNA. Deletion of the N-terminal extension, however, enabled MKT1L to interact with EIF4E6. We speculate that MKT1L may either enhance or inhibit the functions of MKT1-containing complexes.

2020-07-14 | PXD018576 | Pride

The endosomal TbTpr86/TbUsp7/SkpZ (TUS) complex controls surface protein abundance in trypanosomes

Project description:In trypanosomes two deubiquitilating enzymes (DUBs), orthologous to human USP7 and VDU1, control abundance of a cohort of surface proteins, including invariant surface glycoproteins (ISGs). Silencing TbUsp7 partially inhibits endocytosis and invariant surface glycoprotein turnover. S-phase kinase-associated protein 1 (Skp1) has crucial roles in cell cycle progression, transcriptional regulation, signal transduction and other processes in animals and fungi by virtue of being a component of cullin E3 ubiquitin ligases. Unexpectedly, trypanosomes possess multiple Skp1 paralogs, including a divergent paralog designated SkpZ. SkpZ is implicated in suramin-sensitivity and endocytosis and decreases in abundance following TbUsp7 knockdown. SkpZ physically interacts with TbUsp7 and TbTpr86. The latter is a tetratricopeptide-repeat protein also implicated in suramin sensitivity and located close to the flagellar pocket/endosomes, consistent with a role in endocytosis. Further, silencing SkpZ reduced abundance of TbUsp7 and TbTpr86 and many trans-membrane domain surface proteins. Our data indicate that TbTpr, TbUsp7 and SkpZ form the ‘TUS’ complex that regulates abundance of a significant cohort of trypanosome surface proteins.

2025-01-08 | PXD021000 | Pride

A novel essential RNA-binding protein complex in Trypanosoma brucei

Project description:ZC3H5 is an essential cytoplasmic trypanosome protein with a single Cx7Cx5Cx3H zinc finger domain. We here show that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500 and Tb927.7.3040. ZC3H5 interacts directly with Tb927.11.4900, which in turn interacts with Tb927.7.3040. Tb927.11.4900 has a circularly permuted GTPase domain, which is required for the Tb927.7.3040 interaction. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which were enriched in the motif (U/A)UAG(U/A). Tethering of ZC3H5, or other complex components, to a reporter repressed its expression. However, depletion of ZC3H5 in vivo did not increase the abundance of ZC3H5-bound mRNAs; instead, counter-intuitively, there were very minor decreases in a few targets. This was accompanied by marked increases in the abundance of very stable mRNAs encoding ribosomal proteins. Depletion also resulted in an increase in monosomes at the expense of large polysomes, and appearance of a "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex is implicated in quality control during the translation of sub-optimal open reading frames, and that its assembly is regulated by GTP hydrolysis and GTP-GDP exchange.

2020-09-23 | PXD019073 | Pride

Immunoisolation of the trypanosome decapping enzyme ALPH1 delivers a potential subcomplex

Project description:Removal of mRNA 5’ caps primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme DCP2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5´-3´exoribonuclease Xrn1. Kinetoplastida lack DCP2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. The enzyme is composed of a catalytic domain flanked by C-terminal and N-terminal extensions. In our related deposition PXD038550 we analysed the ALPH1 interactome by BioID proximity labelling for the full length protein and truncated versions in order to assign domain specific interactions. We showed that Trypanosoma brucei ALPH1 acts in a complex composed of the trypanosome XRN1 ortholog XRNA and four proteins that are unique to Kinetoplastida. The interactome was validated by reverse experiments targeting T. brucei and T. cruzi XRNA by affinity capture and, additionally, the ALPH1 interacting CMGC-family kinase by BioID. Here we carried out affinity capture with T. brucei and T. cruzi ALPH1, which delivers a potential sub-complex missing the C-terminal interactor XRNA.

2023-06-14 | PXD042322 | Pride

Exploring the vault interactome in the divergent eukaryote Trypanosoma brucei

Project description:Many, but not all Eukaryotes have protein-enclosed compartments called vaults. Vaults are composed of multiple copies of the major vault protein (MVP), symmetrically assembled into a basket like shell. Whilst there is some evidence that these compartments are engaging in mRNA maturation and intriguing structural information of the vault shell is available, the function of vaults remains largely elusive. Here, we set out to explore the vault interactome in the early branching eukaryote Trypanosoma brucei employing a combination of affinity capture and TurboID proximity labelling.

2024-12-12 | PXD056004 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data