Myristoylation Restricts Orientation of GRASP on Membranes and is Critical for Membrane Tethering

Abstract

Mammalian Golgi Reassembly and Stacking Proteins (GRASP) self-interact to organize the Golgi apparatus into an elongated membrane network. Localized at the surfaces of apposed membranes by simultaneous binding of a Golgi-associated protein and insertion of an N-terminal myristate, GRASP proteins tether bilayers through trans complexes. We investigated the mechanism that drives trans pairing, given that unproductive cis interactions within a single membrane are otherwise favored. We established an assay that quantifies GRASP-dependent membrane tethering in vitro and used neutron reflection to determine the structure of the GRASP/membrane complex. In the assay, we substituted a C-terminal His-tag interaction with Ni2+-NTA-functionalized lipid for the normal protein-mediated membrane contact. Hence, fluorescent, NTA-labeled liposomes binds to NTA-doped sparsely-tethered lipid bilayer membranes (stBLMs [1]) only if the added GRASP construct formed trans complexes between the two. While Ni2+ chelation was sufficient to bind non-myristoylated GRASP to membranes, myristoylation increased GRASP tethering efficiency significantly. Neutron reflection showed that non-myristoylated GRASP exhibited no preferential orientation on stBLMs. In contrast, the tethering-competent myristoylated GRASP exhibited a specific orientation, deduced using the GRASP crystal structure [2]. From these neutron results, a model for surface-ligated GRASP could be derived, and docking studies suggest a structure of the membrane-tethering GRASP complex. These results indicate that myristoylation restricts the orientation of GRASP to favor trans complexes for membrane tethering.