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Published March 11, 2021 | Supplemental Material + Accepted Version + Published
Journal Article Open

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication

Abstract

The duplication and ninefold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here, we use hydrogen–deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its subcellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an antiparallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable heterotrimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a sixteenfold reduced binding affinity for Gorab that cannot support centriole duplication. Thus, Gorab dimers at the Golgi exist in equilibrium with Sas6-associated monomers at the centriole to balance Gorab's dual role.

Additional Information

© 2021 Fatalska et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Received: 26 March 2020; Accepted: 11 March 2021; Published: 11 March 2021. We thank all members of the Glover, Dadlez, Dong, and Djinovic labs for their insightful comments and discussions. DMG thanks the Wellcome Trust for an Investigator Award and the National Institute of Neurological Disorders and Stroke of the National Institutes of Health award (R01NS113930). AF and MD were funded in part by the National Science Centre MAESTRO project (UMO-2014/14/ A/NZ1/00306), while the instruments were funded in part by the Centre of Preclinical Research and Technology (POIG.02.02.00-14-024/08-00) and the Foundation of Polish Science TEAM-Tech Core Facility (TEAM TECH CORE FACILITY/2016-2/2) grants. Rotary shadowing EM was performed by the EM Facility of the Vienna BioCenter Core Facilities GmbH (VBCF), member of the Vienna BioCenter (VBC), Austria. Work from GD's lab was supported by grant P28231-B28 from the Austrian Science Fund (FWF). ES is an associated member of the Integrative Structural Biology PhD program funded by the Austrian Science Fund (W-1258 Doktoratskollegs). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Data availability: All data generated or analysed during this study are included in the manuscript and supporting files. Author contributions: Agnieszka Fatalska, Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing, Project initiation, HDX-MS, Generation of deletions and truncations, in vitro binding assays, SEC-MALS; Emma Stepinac, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, EM, ITC, FCS, SEC-MALS; Magdalena Richter, Conceptualization, Investigation, Visualization, Methodology, Writing - review and editing, Generation of truncations and point mutations, in vitro binding assays; Levente Kovacs, Visualization, Methodology, Writing - original draft, Writing - review and editing, Formulation and execution of all in vivo studies, Drosophila genetics, immunostaining and confocal microscopy; Zbigniew Pietras, Formal analysis, Assisted AF in SEC-MALS data collection and processing; Martin Puchinger, Formal analysis, Assisted ES in collection and analysis of FCS data; Gang Dong, Michal Dadlez, Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing; David M Glover, Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing. Competing interests: The authors declare that no competing interests exist.

Attached Files

Published - elife-57241-v4.pdf

Accepted Version - elife-57241-v3.pdf

Supplemental Material - elife-57241-supp-v3.zip

Supplemental Material - elife-57241-transrepform-v4.docx

Supplemental Material - elife-57241-video1.mp4

Supplemental Material - elife-57241-video2.mp4

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Created:
August 20, 2023
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December 22, 2023