The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication
Abstract
The duplication and 9-fold 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 sub-cellular 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 anti-parallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable hetero-trimeric 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 16-fold reduced binding affinity for Gorab that can not support centriole duplication. Thus Gorab dimers at the Golgi exist in equilibrium with Sas-6 associated monomers at the centriole to balance Gorab's dual role.
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All data generated or analysed during this study are included in the manuscript and supporting files.
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Author details
Funding
National Science Centre (MAESTRO (UMO-2014/14/A/NZ1/00306))
- Agnieszka Fatalska
- Michal Dadlez
Wellcome Trust
- Agnieszka Fatalska
- Magdalena Richter
- Levente Kovacs
Austrian Science Fund (P28231-B28)
- Gang Dong
Austrian Science Fund (W-1258 Doktoratskollegs)
- Emma Stepinac
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Fatalska 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.
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Further reading
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- Cell Biology
- Developmental Biology
Long thought to have little relevance to ovarian physiology, the rete ovarii may have a role in follicular dynamics and reproductive health.
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- Cell Biology
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The rete ovarii (RO) is an appendage of the ovary that has been given little attention. Although the RO appears in drawings of the ovary in early versions of Gray’s Anatomy, it disappeared from recent textbooks, and is often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal microscopy, we characterized the fetal development of the RO in the context of the mouse ovary. The RO consists of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that luminal contents flow toward the ovary. Mass spectrometry revealed that the EOR lumen contains secreted proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the extraovarian landscape suggest that it plays an important role in ovary development and homeostasis.