Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

• Published in: The Journal of biological chemistry Vol. 289; no. 14; pp. 9683 - 9691
• Main Authors: Heinrich, Frank, Nanda, Hirsh, Goh, Haw Zan, Bachert, Collin, Lösche, Mathias, Linstedt, Adam D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04-04-2014; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Fluorescence; Golgi; Humans; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Lipoylation; Membrane Bilayer; Membrane Biology; Membrane Fusion; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membrane Trafficking; Membranes, Artificial; Myristic Acid - chemistry; Myristic Acid - metabolism; Neutron Scattering; Protein Structure, Tertiary; Membrane Trafficking; Fluorescence; Golgi; Membrane Bilayer; Membrane Fusion; Neutron Scattering
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.543561

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 Ni2+-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.
Background: An unknown mechanism promotes trans interactions by the GRASP homotypic membrane tethers rather than unproductive cis interactions.
Results: Neutron reflection shows that the myristoylated GRASP domain has a fixed, upright orientation on the membrane incompatible with cis interactions.
Conclusion: Myristoylation restricts the orientation of the protein on the membrane to favor interactions in trans.
Significance: Orientation of membrane proteins is functionally significant and may be regulated by myristoylation.