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.
Highlights
Centrioles are the ninefold symmetrical microtubule arrays found at the core of centrosomes, the bodies that organize cytoplasmic microtubules in interphase and mitosis
A previous study has identified a Golgi-targeting domain region of human Gorab from amino acids 200–277 (Egerer et al, 2015). This region, comprising predominantly a putative coiled-coil sequence, corresponds to aa 246–323 of Drosophila Gorab. To determine whether this region conferred the ability to homodimerize a characteristic of the golgins, we first wished to determine the oligomeric state of Gorab in solution
We expressed N-terminally MBP-tagged Drosophila melanogaster Gorab in Escherichia coli, affinity purified the recombinant protein on amylose resin, and carried out size exclusion chromatography coupled with multiangle light scattering (SECMALS) to determine its molecular mass (Mw) (Figure 1A)
Summary
Centrioles are the ninefold symmetrical microtubule arrays found at the core of centrosomes, the bodies that organize cytoplasmic microtubules in interphase and mitosis. The core components of centrioles and the molecules that regulate their assembly are highly conserved (Brito et al, 2012). The ensuing assembly of a ninefold symmetrical arrangement of Sas dimers provides the structural basis for the ninefold symmetrical cartwheel structure at the procentriole’s core. Sas interacts with Cep135 and in turn with Sas (Drosophila)/CPAP (human), which provides the linkage to centriole microtubules (Hiraki et al, 2007; Jerka-Dziadosz et al, 2010; Kohlmaier et al, 2009; Lin et al, 2013; Roque et al, 2012; Schmidt et al, 2009; Tang et al, 2009)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
