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TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei

Twenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes...

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Autores principales: Ambekar, Sushma V., Beck, Josh R., Mair, Gunnar R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9601220/
https://www.ncbi.nlm.nih.gov/pubmed/36040030
http://dx.doi.org/10.1128/mbio.01815-22
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author Ambekar, Sushma V.
Beck, Josh R.
Mair, Gunnar R.
author_facet Ambekar, Sushma V.
Beck, Josh R.
Mair, Gunnar R.
author_sort Ambekar, Sushma V.
collection PubMed
description Twenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups — Nup176, 269, 335, 390, and 434 — are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position.
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spelling pubmed-96012202022-10-27 TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei Ambekar, Sushma V. Beck, Josh R. Mair, Gunnar R. mBio Research Article Twenty years since the publication of the Plasmodium falciparum and P. berghei genomes one-third of their protein-coding genes still lack functional annotation. In the absence of sequence and structural homology, protein-protein interactions can facilitate functional prediction of such orphan genes by mapping protein complexes in their natural cellular environment. The Plasmodium nuclear pore complex (NPC) is a case in point: it remains poorly defined; its constituents lack conservation with the 30+ proteins described in the NPC of many opisthokonts, a clade of eukaryotes that includes fungi and animals, but not Plasmodium. Here, we developed a labeling methodology based on TurboID fusion proteins, which allows visualization of the P. berghei NPC and facilitates the identification of its components. Following affinity purification and mass spectrometry, we identified 4 known nucleoporins (Nups) (138, 205, 221, and the bait 313), and verify interaction with the putative phenylalanine-glycine (FG) Nup637; we assigned 5 proteins lacking annotation (and therefore meaningful homology with proteins outside the genus) to the NPC, which is confirmed by green fluorescent protein (GFP) tagging. Based on gene deletion attempts, all new Nups — Nup176, 269, 335, 390, and 434 — are essential to parasite survival. They lack primary sequence homology with proteins outside the Plasmodium genus; albeit 2 incorporate short domains with structural homology to human Nup155 and yeast Nup157, and the condensin SMC (Structural Maintenance Of Chromosomes 4). The protocols developed here showcase the power of proximity labeling for elucidating protein complex composition and annotation of taxonomically restricted genes in Plasmodium. It opens the door to exploring the function of the Plasmodium NPC and understanding its evolutionary position. American Society for Microbiology 2022-08-30 /pmc/articles/PMC9601220/ /pubmed/36040030 http://dx.doi.org/10.1128/mbio.01815-22 Text en Copyright © 2022 Ambekar et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Ambekar, Sushma V.
Beck, Josh R.
Mair, Gunnar R.
TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei
title TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei
title_full TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei
title_fullStr TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei
title_full_unstemmed TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei
title_short TurboID Identification of Evolutionarily Divergent Components of the Nuclear Pore Complex in the Malaria Model Plasmodium berghei
title_sort turboid identification of evolutionarily divergent components of the nuclear pore complex in the malaria model plasmodium berghei
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9601220/
https://www.ncbi.nlm.nih.gov/pubmed/36040030
http://dx.doi.org/10.1128/mbio.01815-22
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