Dual anchoring of the GRASP membrane tether promotes trans pairing.
GRASP proteins share an N-terminal GRASP domain and mediate homotypic tethering of Golgi cisternae to form extended Golgi ribbons. The golgin GM130 is thought to bind the C-terminal side of the GRASP domain to recruit GRASP65 onto the Golgi whereas stable membrane association appears to also depend on anchoring of the N terminus by myristoylation. Here, we examine the nature of the GM130/GRASP65 interaction and test whether the dual membrane contacts of the GRASP domain have a role in tethering beyond membrane recruitment. GM130 was found to contain a C-terminal PDZ ligand that binds the putative groove of the second PDZ-like domain in GRASP65. To test tethering activity independent of targeting, we took advantage of a tethering assay carried out on the mitochondrial membrane in which the GRASP membrane attachment points were individually or simultaneously substituted with mitochondrially targeted transmembrane sequences. N-terminally anchored constructs tethered only if the C terminus was also anchored; and likewise, C-terminally anchored constructs tethered only if the N terminus was anchored. One explanation for the role of this dual anchoring is that it orients the GRASP domain to prevent cis interactions within the same membrane thereby favoring trans interactions between adjacent membranes. Indeed, singly anchored GRASP constructs, although nonfunctional in tethering, interacted with one another and also bound and inhibited dually anchored constructs. This work thus elucidates the GM130/GRASP65 interaction and supports a novel orientation-based model of membrane tether regulation in which dual membrane contact orients the tethering interaction interface to favor trans over cis interactions.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Collin
C
Bachert
Adam D
AD
Linstedt
D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D008954:Models, Biological; D017434:Protein Structure, Tertiary
D001324:Autoantigens; C108705:GORASP1 protein, human; D000077614:Golgi Matrix Proteins; C097741:Golgin subfamily A member 2; D008565:Membrane Proteins