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.

Vimentin intermediate filaments provide structural stability to the mammalian Golgi apparatus

“…It is interesting to note that GORAB oligomerization is necessary for binding to vimentin IFs, and it is also required for the formation of TGN-associated GORAB domains involved in COPI vesicle traffic (Witkos et al, 2019). Hence, GORAB oligomerization may control the various functions of the protein, as proposed by Fatalaska et al, who found that centriolar GORAB is present as a monomer, whereas it is a dimer at the TGN (Fatalska et al, 2021). Interestingly, the K190 deletion, a causative mutation in GO that disrupts GORAB olgomerisation, abolishes both TGN domain formation and IF binding, raising the possibility that IF binding could also be disrupted in GO.…”

“…GORAB is also present at the centrioles, where it binds the cartwheel protein SAS-6 (Kovacs et al, 2018). GORAB binds SAS-6 as a monomer, whereas at the TGN it is present as a dimer (Fatalska et al, 2021). The function of GORAB at the centriole is unclear, but it may act as a physical scaffold, analogous to its proposed function at the TGN.…”

The factory, the antenna and the scaffold: the three-way interplay between the Golgi, cilium and extracellular matrix underlying tissue function

“…However, there are suggestions they interact. In Drosophila , monomeric Gorab localises to centrioles ( Fatalska et al, 2021 ; Kovacs et al, 2018 ), and the cilia of gorab null sensory neurons have disorganised ciliary rootlets and disrupted ninefold radial symmetry ( Kovacs et al, 2018 ). In these studies, Gorab function in Golgi trafficking and ciliogenesis can be separated suggesting the compartments are not directly interacting.…”

Nine‐fold symmetry of centriole: The joint efforts of its core proteins

“…[ 130 ] In vitro experiment showed that Gorab binds to the C‐terminal region of Sas6 dimer via its C‐terminal part, and its N terminus extends away from Sas6. [ 130,131 ]…”