A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex

Choi, Charles Paul and Moon, Andy Seong and Back, Peter Sungmin and Jami‐Alahmadi, Yasaman and Vashisht, Ajay Amar and Wohlschlegel, James Akira and Bradley, Peter John and Coppens, Isabelle (2019) A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex. PLOS Biology, 17 (10). e3000475. ISSN 1545-7885

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Abstract

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite’s plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein–protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein–protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.

Item Type: Article
Subjects: Souths Book > Biological Science
Depositing User: Unnamed user with email support@southsbook.com
Date Deposited: 24 Jan 2023 08:01
Last Modified: 22 May 2024 09:55
URI: http://research.europeanlibrarypress.com/id/eprint/63

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