Dual Anchoring of the GRASP Membrane Tether Promotes trans Pairing

Adam D. Linstedt,Collin Bachert

doi:10.1074/jbc.m110.116129

Abstract

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.

Highlights

GRASP65 PDZ2 Binds a PDZ Ligand in GM130 for Membrane Recruitment—Conventional PDZ ligand sequences have been classified on the basis of residue positioning relative to the C-terminal residue termed P0, with the residue at PϪ2 considered most important [15]
It is likely that the expressed nonmyristoylated form in these experiments inhibited the endogenous GRASP65 recruited to the mitochondria as well. These experiments demonstrate the importance of dual anchoring of the GRASP domain and strongly suggest that it orients the molecule to favor interactions in trans. These studies support a model (Fig. 9) in which GRASP65 is recruited onto the Golgi by GM130 whereupon GRASP65 membrane attachment is stabilized by insertion of its N-terminal myristic acid
These two contact points, membrane binding by the myristic acid and GM130 binding, orient the GRASP65 homotypic binding interface in such a way that it can interact in trans but not in cis

Summary

Dual Anchoring of the GRASP Membrane Tether Promotes trans Pairing*

The GLGF motif is present near the N terminus of PDZ domains just prior to the second ␤-strand, which forms one side of the binding pocket [8], whereas the GLGF motif of GRASP65 mutated in previous work is near the C terminus just after the predicted sixth ␤-strand. It is not known whether anchoring the C-terminal side of PDZ1, i.e. Anchoring of GRASP Membrane Tether establishing a dually anchored PDZ1 domain, plays a functional role in tethering beyond targeting the complex to the membrane. We further show that this interaction is sufficient to recruit GRASP65 to membranes and regulates its ability to tether membranes in trans by anchoring both ends of the molecule possibly geometrically restricting it to prevent cis interactions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION