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.

Summary Extended synaptotagmins (E-Syts) localize at membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane to mediate inter-membrane lipid transfer and control plasma membrane lipid homeostasis. All known E-Syts contain an N-terminal transmembrane (TM) hairpin, a central synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain, and three or five C2 domains at their C termini. Here we report an uncharacterized E-Syt from the protist parasite Trypanosoma brucei , namely, TbE-Syt. TbE-Syt contains only two C2 domains (C2A and C2B), making it the shortest E-Syt known by now. We determined a 1.5-Å-resolution crystal structure of TbE-Syt-C2B and revealed that it binds lipids via both Ca 2+ - and PI(4,5)P 2 -dependent means. In contrast, TbE-Syt-C2A lacks the Ca 2+ -binding site but may still interact with lipids via a basic surface patch. Our studies suggest a mechanism for how TbE-Syt tethers the ER membrane tightly to the plasma membrane to transfer lipids between the two organelles.