Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

ABSTRACT The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell i...

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Autores principales:	Allan L. Chen, Elliot W. Kim, Justin Y. Toh, Ajay A. Vashisht, Andrew Q. Rashoff, Christina Van, Amy S. Huang, Andy S. Moon, Hannah N. Bell, Laurent A. Bentolila, James A. Wohlschlegel, Peter J. Bradley
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/73c36f7817104660909eec325ebdd9d6
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spelling	oai:doaj.org-article:73c36f7817104660909eec325ebdd9d62021-11-15T15:41:19ZNovel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID10.1128/mBio.02357-142150-7511https://doaj.org/article/73c36f7817104660909eec325ebdd9d62015-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02357-14https://doaj.org/toc/2150-7511ABSTRACT The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. IMPORTANCE The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.Allan L. ChenElliot W. KimJustin Y. TohAjay A. VashishtAndrew Q. RashoffChristina VanAmy S. HuangAndy S. MoonHannah N. BellLaurent A. BentolilaJames A. WohlschlegelPeter J. BradleyAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 1 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Allan L. Chen Elliot W. Kim Justin Y. Toh Ajay A. Vashisht Andrew Q. Rashoff Christina Van Amy S. Huang Andy S. Moon Hannah N. Bell Laurent A. Bentolila James A. Wohlschlegel Peter J. Bradley Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID
description	ABSTRACT The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. IMPORTANCE The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.
format	article
author	Allan L. Chen Elliot W. Kim Justin Y. Toh Ajay A. Vashisht Andrew Q. Rashoff Christina Van Amy S. Huang Andy S. Moon Hannah N. Bell Laurent A. Bentolila James A. Wohlschlegel Peter J. Bradley
author_facet	Allan L. Chen Elliot W. Kim Justin Y. Toh Ajay A. Vashisht Andrew Q. Rashoff Christina Van Amy S. Huang Andy S. Moon Hannah N. Bell Laurent A. Bentolila James A. Wohlschlegel Peter J. Bradley
author_sort	Allan L. Chen
title	Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID
title_short	Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID
title_full	Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID
title_fullStr	Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID
title_full_unstemmed	Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID
title_sort	novel components of the toxoplasma inner membrane complex revealed by bioid
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/73c36f7817104660909eec325ebdd9d6
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Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

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