The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication
- University of Cambridge, United Kingdom
- Medical University of Vienna, Austria
- Institute of Biochemistry and Biophysics PAS, Poland
- University of Vienna, Austria
- Institute of Biochemistry and Biophysics PAN, Poland
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.
Article and author information
Author details
Agnieszka Fatalska
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
Distal appendages are ninefold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for the formation of the primary cilium, by regulating at least four critical steps: preciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here, we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in the RAB34+ vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
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- Cell Biology
- Medicine
Background:
Pulmonary vascular remodeling is a progressive pathological process characterized by functional alterations within pulmonary artery smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs). Mechanisms driving the transition to a diseased phenotype remain elusive.
Methods:
We combined transcriptomic and proteomic profiling with phenotypic characterization of source-matched cells from healthy controls and individuals with idiopathic pulmonary arterial hypertension (IPAH). Bidirectional cellular crosstalk was examined using direct and indirect co-culture models, and phenotypic responses were assessed via transcriptome analysis.
Results:
PASMC and PAAF undergo distinct phenotypic shifts during pulmonary vascular remodeling, with limited shared features, such as reduced mitochondrial content and hyperpolarization. IPAH-PASMC exhibit increased glycosaminoglycan production and downregulation of contractile machinery, while IPAH-PAAF display a hyperproliferative phenotype. We identified alterations in extracellular matrix components, including laminin and collagen, alongside pentraxin-3 and hepatocyte growth factor, as potential regulators of PASMC phenotypic transitions mediated by PAAF.
Conclusions:
While PASMCs and PAAFs retain their core cellular identities, they acquire distinct disease-associated states. These findings provide new insights into the dynamic interplay of pulmonary vascular mesenchymal cells in disease pathogenesis.
Funding:
This work was supported by Cardio-Pulmonary Institute EXC 2026 390649896 (GK) and Austrian Science Fund (FWF) grant I 4651-B (SC).
