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Probing the trypanosome nuclear pore by TurboID proximity labelling – Part2

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Trypanosoma Brucei

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Trypanosomiasis

SUBMITTER: Martin Zoltner

LAB HEAD: Dr. Martin Zoltner

PROVIDER: PXD055934 | Pride | 2024-09-20

REPOSITORIES: Pride

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			Action	DRS
	MEX67.combined.txt.rar	Txt
	MEX67_1.raw	Raw
	MEX67_2.raw	Raw
	MEX67_3.raw	Raw
	NUP75C_1.raw	Raw

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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.

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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

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2022-10-15 | PXD037289 | Pride

Probing the trypanosome nuclear pore by TurboID proximity labelling

Project description:Previous BioID experiments targeting the nucleoporin (NUP) NUP158 and the nuclear transport factor MEX67 indicated (PMID: 36410438; PXD031245) 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. Here we extended this approach to target NUPs NUP110, NUP76 and NUP96 with a TurboID-HA tandem tag fused to both, the N and C-terminus of each respective target protein. While the central NUP96 biotinylated most nuclear pore proteins with the marked exception of the nuclear basket, NUP158, NUP110 and NUP76 caused biotinylation of specific sub-complexes of the pore and Mex67 labelled NUPs lining the pore channel.

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2023-06-14 | PXD038550 | Pride

A novel essential RNA-binding protein complex in Trypanosoma brucei

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