Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 May 21;285(21):16294-301.
doi: 10.1074/jbc.M110.116129. Epub 2010 Mar 12.

Dual anchoring of the GRASP membrane tether promotes trans pairing

Collin Bachert  1 , Adam D Linstedt
Affiliations

Dual anchoring of the GRASP membrane tether promotes trans pairing

Collin Bachert et al. J Biol Chem. .

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.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
GM130 contains a C-terminal PDZ ligand. A, schematic of the C termini of GM130 through evolution from its first appearance in Drosophila melanogaster to Homo sapiens. B, purified myristoylated G65-His (2 μg) incubated with the GST-tagged GM130 C terminus with or without an appended arginine (2 μg). Recovery on glutathione-agarose beads was determined by immunoblotting (anti-His) and staining with Ponceau S. Binding was quantified (n = 3, ±S.E. (error bars)).
FIGURE 2.
FIGURE 2.
GM130 recruits GRASP65 to membranes using a C-terminal ligand. Cells were transfected with mitochondrially targeted GM130 C terminus (T20-GFP-GM130Cterm) without (A–C) or with an additional arginine (E–G). The cells were treated with brefeldin A to disperse the Golgi and visualized to detect GFP fluorescence and endogenous GRASP65 using an anti-GRASP65 antibody. The levels of GRASP65 recruitment are also indicated by representations of the co-localized pixels (D and H). Scale bar, 10 μm.
FIGURE 3.
FIGURE 3.
C terminus of GM130 binds PDZ2 of GRASP65. Purified, myristoylated G65-His (2 μg) as wild type or with mutations in the predicted binding pocket of PDZ1 (LL58,59AA) or PDZ2 (LI152,153AA) was incubated with GST-GM130Cterm or GST (2 μg). Recovery on glutathione-agarose beads was determined by immunoblotting (anti-His) and staining with Ponceau S. Binding was quantified (n = 3, ±S.E. (error bars)).
FIGURE 4.
FIGURE 4.
GRASP65 is recruited to the Golgi by the PDZ2/GM130 ligand interaction. Cells were transfected with the wild type G65-myc (A–C), the PDZ1 mutant G65L58A/L59A-myc (D–F), or the PDZ2 mutant G65L152A/I153A-myc (G–I) and stained using antibodies against myc and GPP130 (to stain the Golgi). Scale bar, 10 μm.
FIGURE 5.
FIGURE 5.
C-terminal anchoring of GRASP65 is required for tethering of mitochondria. Cells were transfected with the indicated mitochondrially targeted constructs and visualized using GFP fluorescence followed by quantification of mitochondrial clustering with radial profile analysis (n = 3, 15 cells each trial, ±S.E.). Scale bar, 10 μm.
FIGURE 6.
FIGURE 6.
Singly anchored GRASP65 is binding-competent. Cells co-transfected with the indicated myc and GFP-tagged mitochondrial GRASP65 constructs were subjected to immunoprecipitation with anti-myc antibodies followed by immunoblotting with anti GRASP65 (G65, to detect myc-tagged constructs) or anti-GFP antibodies (to detect co-precipitation). Constructs were T20-G65 with either a myc or GFP tag followed by either a C-terminal anchor (ActA) or a stop codon (Stop). Co-transfection with an empty vector was used as a negative control (Empty). Loading control is 10% of total extract incubated with beads. Binding was quantified as the ratio of the amount of co-precipitated GFP-tagged protein to recovered myc-tagged protein (n = 3, ±S.E. (error bars)).
FIGURE 7.
FIGURE 7.
Singly anchored GRASP65 inhibits the tethering of mitochondria by doubly anchored GRASP65. HeLa cells co-transfected with the indicated mitochondrially targeted GRASP65 constructs were visualized using GFP fluorescence and myc staining, and clustering was quantified using radial profile analysis (n = 3, 15 cells each trial, ±S.E. (error bars)). Shown are two dually anchored versions (A–D), a dual with a single (E–H), two singly anchored versions (I–L), and a dual with a negative control (M–P). Scale bar, 10 μm. Direct comparison of the fluorescence within radius 50 pixels is also shown (Q).
FIGURE 8.
FIGURE 8.
Nonmyristoylated GRASP65 is recruited to mitochondrial membranes and inhibits tethering by endogenous GRASP65. HeLa cells co-transfected with the mitochondrial GM130 C terminus (T20-GFP-GM130Cterm) and either G65-myc (A–D) or the nonmyristoylated G65G2A-myc (E–H) were visualized using GFP fluorescence and myc staining, and clustering was quantified using radial profile analysis (n = 3, 15 cells each trial, ±S.E.). Scale bar, 10 μm.
FIGURE 9.
FIGURE 9.
Model of trans pair disassembly by membrane rearrangement upon fusion. Note that the interaction interface is oriented by the combined myristic acid and golgin attachment of the GRASP domain in such a way that it prevents cis interactions before and after fusion.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Waters M. G., Pfeffer S. R. (1999) Curr. Opin. Cell Biol. 11, 453–459 - PubMed
    1. Whiteheart S. W., Schraw T., Matveeva E. A. (2001) Int. Rev. Cytol. 207, 71–112 - PubMed
    1. Puthenveedu M. A., Bachert C., Puri S., Lanni F., Linstedt A. D. (2006) Nat. Cell Biol. 8, 238–248 - PubMed
    1. Feinstein T. N., Linstedt A. D. (2008) Mol. Biol. Cell 19, 2696–2707 - PMC - PubMed
    1. Wang Y., Satoh A., Warren G. (2005) J. Biol. Chem. 280, 4921–4928 - PMC - PubMed

Publication types

LinkOut - more resources

-