Integrative modeling reveals the molecular architecture of the intraflagellar transport A (IFT-A) complex
- The University of Texas at Austin, United States
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- Human Technopole, Italy
Abstract
Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.
Data availability
Mass spectrometry proteomics data was deposited in the MassIVE/ProteomeXchange database (113) under accession number PXD032818. Cryo-tomography data was deposited in the Electron Microscopy Data Bank (114) under accession number EMD-26791. IFT-A models were deposited in the PDB-Dev database (115) as well as on Zenodo at doi: 10.5281/zenodo.7222413, along with additional supporting materials, including integrative modeling data and code.
Article and author information
Author details
Funding
National Science Foundation (2019238253)
- Caitlyn L McCafferty
National Institute of General Medical Sciences (R35GM122480)
- Edward M Marcotte
National Institute of General Medical Sciences (R35GM138348)
- David W Taylor
National Institute of Child Health and Human Development (HD085901)
- John B Wallingford
- Edward M Marcotte
Army Research Office (W911NF-12-1-0390)
- Edward M Marcotte
Welch Foundation (F-1515)
- Edward M Marcotte
Welch Foundation (F-1938)
- David W Taylor
Max Planck Society
- Mareike A Jordan
- Gaia Pigino
Cancer Prevention and Research Institute of Texas (RR160088)
- David W Taylor
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, McCafferty et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,221
- views
-
- 329
- downloads
-
- 23
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Integrative modeling reveals the molecular architecture of the intraflagellar transport A (IFT-A) complex
eLife 11:e81977.
https://doi.org/10.7554/eLife.81977
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3’-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.
-
- Biochemistry and Chemical Biology
- Genetics and Genomics
Yerba mate (YM, Ilex paraguariensis) is an economically important crop marketed for the elaboration of mate, the third-most widely consumed caffeine-containing infusion worldwide. Here, we report the first genome assembly of this species, which has a total length of 1.06 Gb and contains 53,390 protein-coding genes. Comparative analyses revealed that the large YM genome size is partly due to a whole-genome duplication (Ip-α) during the early evolutionary history of Ilex, in addition to the hexaploidization event (γ) shared by core eudicots. Characterization of the genome allowed us to clone the genes encoding methyltransferase enzymes that catalyse multiple reactions required for caffeine production. To our surprise, this species has converged upon a different biochemical pathway compared to that of coffee and tea. In order to gain insight into the structural basis for the convergent enzyme activities, we obtained a crystal structure for the terminal enzyme in the pathway that forms caffeine. The structure reveals that convergent solutions have evolved for substrate positioning because different amino acid residues facilitate a different substrate orientation such that efficient methylation occurs in the independently evolved enzymes in YM and coffee. While our results show phylogenomic constraint limits the genes coopted for convergence of caffeine biosynthesis, the X-ray diffraction data suggest structural constraints are minimal for the convergent evolution of individual reactions.
