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Organelle tethering by a homotypic PDZ interaction underlies formation of the Golgi membrane network.

Sengupta D, Truschel S, Bachert C, Linstedt AD - J. Cell Biol. (2009)

Bottom Line: Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction.Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans.Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

ABSTRACT
Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

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Related in: MedlinePlus

Membrane insertion of the GRASP65 N terminus is required. Schematic diagram of the constructs (A). HeLa cells expressing G65G2A-GFP-ActA (B–D and K), T20-G65-GFP-ActA (E–G and L), or T20-GFP-ActA (H–J and M) were BFA treated and analyzed using Mitotracker (red), GFP fluorescence (green), and radial profile plots. Bar, 10 µm.
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fig8: Membrane insertion of the GRASP65 N terminus is required. Schematic diagram of the constructs (A). HeLa cells expressing G65G2A-GFP-ActA (B–D and K), T20-G65-GFP-ActA (E–G and L), or T20-GFP-ActA (H–J and M) were BFA treated and analyzed using Mitotracker (red), GFP fluorescence (green), and radial profile plots. Bar, 10 µm.

Mentions: GRASP65 is myristoylated at its N terminus and this modification, together with GM130 binding, mediates GRASP65 localization to the Golgi membrane (Barr et al., 1998; Puthenveedu et al., 2006). Although the G65-GFP-ActA construct is expected to be N-myristoylated, the construct also has a C-terminal transmembrane anchor that would presumably make its membrane integration independent of myristoylation. Indeed, when the construct was modified by alanine substitution of the glycine acceptor site to prevent its myristoylation, the resulting construct, G65G2A-GFP-ActA, was targeted to mitochondria as indicated by colocalization with Mitotracker staining (Fig. 8, A–D). Unexpectedly, however, G65G2A-GFP-ActA failed to cause mitochondrial clustering as verified using radial profile analysis (Fig. 8 K) and also expression in mfn−/− cells (unpublished data). In light of the fact that previous studies of GRASP65 oligomerization-induced crossbridging were performed in vitro using nonmyristoylated protein (Wang et al., 2003, 2005), this result underscores the importance of demonstrating organelle tethering by GRASP65 in vivo.


Organelle tethering by a homotypic PDZ interaction underlies formation of the Golgi membrane network.

Sengupta D, Truschel S, Bachert C, Linstedt AD - J. Cell Biol. (2009)

Membrane insertion of the GRASP65 N terminus is required. Schematic diagram of the constructs (A). HeLa cells expressing G65G2A-GFP-ActA (B–D and K), T20-G65-GFP-ActA (E–G and L), or T20-GFP-ActA (H–J and M) were BFA treated and analyzed using Mitotracker (red), GFP fluorescence (green), and radial profile plots. Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712994&req=5

fig8: Membrane insertion of the GRASP65 N terminus is required. Schematic diagram of the constructs (A). HeLa cells expressing G65G2A-GFP-ActA (B–D and K), T20-G65-GFP-ActA (E–G and L), or T20-GFP-ActA (H–J and M) were BFA treated and analyzed using Mitotracker (red), GFP fluorescence (green), and radial profile plots. Bar, 10 µm.
Mentions: GRASP65 is myristoylated at its N terminus and this modification, together with GM130 binding, mediates GRASP65 localization to the Golgi membrane (Barr et al., 1998; Puthenveedu et al., 2006). Although the G65-GFP-ActA construct is expected to be N-myristoylated, the construct also has a C-terminal transmembrane anchor that would presumably make its membrane integration independent of myristoylation. Indeed, when the construct was modified by alanine substitution of the glycine acceptor site to prevent its myristoylation, the resulting construct, G65G2A-GFP-ActA, was targeted to mitochondria as indicated by colocalization with Mitotracker staining (Fig. 8, A–D). Unexpectedly, however, G65G2A-GFP-ActA failed to cause mitochondrial clustering as verified using radial profile analysis (Fig. 8 K) and also expression in mfn−/− cells (unpublished data). In light of the fact that previous studies of GRASP65 oligomerization-induced crossbridging were performed in vitro using nonmyristoylated protein (Wang et al., 2003, 2005), this result underscores the importance of demonstrating organelle tethering by GRASP65 in vivo.

Bottom Line: Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction.Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans.Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

ABSTRACT
Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

Show MeSH
Related in: MedlinePlus