RESUMO
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 Ca2+- and PI(4,5)P2-dependent means. In contrast, TbE-Syt-C2A lacks the Ca2+-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.
RESUMO
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.