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

Autor: Choi CP; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, United States of America., Moon AS; Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, United States of America., Back PS; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, United States of America., Jami-Alahmadi Y; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, United States of America., Vashisht AA; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, United States of America., Wohlschlegel JA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, United States of America.; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, United States of America., Bradley PJ; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, United States of America.; Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, United States of America.
Jazyk: angličtina
Zdroj: PLoS biology [PLoS Biol] 2019 Oct 04; Vol. 17 (10), pp. e3000475. Date of Electronic Publication: 2019 Oct 04 (Print Publication: 2019).
DOI: 10.1371/journal.pbio.3000475
Abstrakt: 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.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
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