title: Myristoylation restricts orientation of the GRASP domain on membranes and promotes membrane tethering.
abstract: The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni(2+)-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering.
authors: From the Departments of Physics.
authors: Frank
authors: F
authors: Heinrich
authors: Hirsh
authors: H
authors: Nanda
authors: Haw Zan
authors: HZ
authors: Goh
authors: Collin
authors: C
authors: Bachert
authors: Mathias
authors: M
authors: Lösche
authors: Adam D
authors: AD
authors: Linstedt
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D054878:Lipoylation; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008567:Membranes, Artificial; D019814:Myristic Acid / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D002352:Carrier Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D008567:Membranes, Artificial; C410230:tamalin protein, human; D019814:Myristic Acid
keywords: Fluorescence; Golgi; Membrane Bilayer; Membrane Fusion; Membrane Trafficking; Neutron Scattering