Dual Anchoring of the GRASP Membrane Tether Promotes trans Pairing

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.The tethering of membranes prior to SNARE 2 -mediated fusion is accomplished by factors present in each membrane that interact to form a bridge thereby increasing the fidelity and efficiency of membrane fusion (1). The interaction can be heterotypic, such as vesicle fusion with a target organelle, or homotypic, as is the case when identical membranes fuse. In heterotypic tethering, each membrane contributes a distinct partner to form a heteromeric-tethering complex. After fusion, the complex is presumably disassembled, and one partner, if still membrane-associated, is sorted from the other in the plane of the membrane and packaged into vesicles for recycling. Disassembly and sorting are needed because interactions in cis between the tether partners would prevent further rounds of tethering just as cis SNARE interactions block membrane fusion (2). In homotypic tethering, each membrane may contribute distinct partners that bind to one another or identical partners that self-associate but because the membranes are identical each has a full complement of the tethering components, and this state is also maintained after fusion. Thus, for homotypic tethering, mechanisms other than sorting and recycling are needed to prevent cis complex formation.In higher eukaryotes, the juxtanuclear Golgi membrane network, or Golgi ribbon, is...

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

“…5 The background of nonspecific protein binding, quantified with myrG55 on DOPC stBLMs devoid of Ni 2ϩ -NTA-DGS, was Ͻ10% of the signal with Ni 2ϩ -NTA-DGS. This is consistent with the relatively weak affinity of myristate for the membrane, K d ϳ100 mol/ liter (32), and with the inability of myristoylation alone to localize the GRASP proteins to membranes in cells (13,15).…”

“…The relative positions of the internal ligand and the PDZ1 binding groove on the molecule (11) suggest that GRASP orientation on the membrane could provide a mechanism that favors homotypic interactions in trans over those in cis (15). However, for such a mechanism to work, the orientation of the protein with respect to the membrane needs to be tightly controlled, raising the possibility that N-myristoylation, as a second anchor point for the protein on the membrane, could set this orientation.…”

“…Stable, singly anchored versions of GRASP proteins have been created using transmembrane domains but, when expressed in cells, they fail to tether, whereas similar dually anchored constructs retain full activity (12,15). The singly anchored constructs are recovered in complexes, suggesting that they form cis interactions within the same membrane that are unproductive for tethering (15).…”

Membrane adhesion dictates Golgi stacking and cisternal morphology

“…The GRASP proteins bind to the Golgins (note that we use the term "Golgin" in a limited sense in this article to refer either to GM130 or Golgin45) (10,11). An appealing mechanism for intercisternal adhesion has been proposed for the GRASP proteins based on X-ray crystallography and biochemistry that involves PSD-95, Dlg1, Zo-1 domain-dependent homo-oligomerization in trans (13)(14)(15). In recent years, with the advent of RNAi-based technologies, knock-down studies have broadly confirmed a role for GRASP proteins and Golgins in controlling Golgi morphology but have not agreed with each other on many notable details, leaving the field in a somewhat confused and conflictory state (10,(15)(16)(17)(18)(19)(20).…”

Structural Insight into Golgi Membrane Stacking by GRASP65 and GRASP55 Proteins

“…In an effort to identify components required for post-mitotic reassembly of Golgi cisternae, a peripheral Golgi protein termed GRASP65 (Golgi reassembly stacking protein of 65 kDa) was identified (5). GRASP65 localizes to the cis-Golgi through its N-terminal myristoylation and interaction with GM130, a cis-Golgi marker (6,7). Modification of GRASP65 on mitotic Golgi fragments by N-ethylmaleimide in vitro and the microinjection of antibodies against GRASP65 into mitotic cells inhibit the stacking of reformed Golgi cisternae (5,8).…”