Department of Genetics, University of Cambridge, Cambridge, United Kingdom; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
Emma Stepinac
Department of Medical Biochemistry, Max Perutz Labs, Medical University of Vienna, Vienna, Austria
Magdalena Richter
Department of Genetics, University of Cambridge, Cambridge, United Kingdom
Levente Kovacs
Department of Genetics, University of Cambridge, Cambridge, United Kingdom; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
Zbigniew Pietras
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
Martin Puchinger
Department of Structural and Computational Biology, Max Perutz Labs, University of Vienna, Vienna, Austria
Department of Medical Biochemistry, Max Perutz Labs, Medical University of Vienna, Vienna, Austria
Michal Dadlez
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
David M Glover
Department of Genetics, University of Cambridge, Cambridge, United Kingdom; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
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.
