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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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Endogenous GRASP65 is recruited to mitochondria by GM130. HeLa cells expressing T20-GFP (A–D), T20-GFP-GM130Cterm (E–H), or T20-GFP-GM130CΔ10 (J–M) were BFA treated and processed to reveal GFP fluorescence, GRASP65 staining, merged images and, from single optical sections, representations of the colocalized pixels. Bar, 10 µm. GRASP65 recruitment (N) was assayed by determining the fraction of total GFP-positive pixels in single optical sections (chosen to maximize mitochondrial representation) that colocalized with GRASP65 staining (n = 3, ±SEM, >15 cells/experiment; *, P < 0.0001).
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fig5: Endogenous GRASP65 is recruited to mitochondria by GM130. HeLa cells expressing T20-GFP (A–D), T20-GFP-GM130Cterm (E–H), or T20-GFP-GM130CΔ10 (J–M) were BFA treated and processed to reveal GFP fluorescence, GRASP65 staining, merged images and, from single optical sections, representations of the colocalized pixels. Bar, 10 µm. GRASP65 recruitment (N) was assayed by determining the fraction of total GFP-positive pixels in single optical sections (chosen to maximize mitochondrial representation) that colocalized with GRASP65 staining (n = 3, ±SEM, >15 cells/experiment; *, P < 0.0001).

Mentions: The absence of clustering by T20-GFP-GM130CΔ10 strongly suggests that T20-GFP-GM130Cterm clusters mitochondria by recruiting endogenous GRASP65. As a test, GRASP65 localization was determined. In BFA-treated cells GRASP65 is known to be principally associated with remnant membrane structures localized adjacent to distributed ER exit sites (Seemann et al., 2000; Ward et al., 2001). Consistent with this localization, GRASP65 was present in dispersed punctate structures in cells expressing T20-GFP, which itself was localized to filamentous mitochondria (Fig. 5, A–D). In striking contrast, GRASP65 localization was largely juxtanuclear in cells expressing T20-GFP-GM130Cterm, and it was clearly evident on the clustered mitochondria (Fig. 5, E–H). In the case of cells expressing T20-GFP-GM130CΔ10, GRASP65 retained the control BFA remnant pattern and was distinct from the filamentous mitochondria (Fig. 5, J–M). GRASP65 coincidence with the GFP constructs was analyzed on a pixel-by-pixel basis and the quantified results indicated significant specific recruitment of endogenous GRASP65 to mitochondria by T20-GFP-GM130Cterm (Fig. 5 N). Thus, the GM130 C terminus recruited endogenous GRASP65 to mitochondria and this induced their clustering. Because GM130 is required for GRASP65 localization to Golgi membranes (Puthenveedu et al., 2006; Kodani and Sutterlin, 2008) and because the GRASP65 binding site in GM130 is required for GM130-dependent Golgi linking (Puthenveedu et al., 2006), these results strongly suggest that GM130 links Golgi ribbons by recruiting GRASP65, which in turn is sufficient to link membranes. Additionally, the results show that endogenous levels of GRASP65 are sufficient to induce mitochondrial clustering, ruling out concerns regarding overexpression of exogenous constructs.


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)

Endogenous GRASP65 is recruited to mitochondria by GM130. HeLa cells expressing T20-GFP (A–D), T20-GFP-GM130Cterm (E–H), or T20-GFP-GM130CΔ10 (J–M) were BFA treated and processed to reveal GFP fluorescence, GRASP65 staining, merged images and, from single optical sections, representations of the colocalized pixels. Bar, 10 µm. GRASP65 recruitment (N) was assayed by determining the fraction of total GFP-positive pixels in single optical sections (chosen to maximize mitochondrial representation) that colocalized with GRASP65 staining (n = 3, ±SEM, >15 cells/experiment; *, P < 0.0001).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Endogenous GRASP65 is recruited to mitochondria by GM130. HeLa cells expressing T20-GFP (A–D), T20-GFP-GM130Cterm (E–H), or T20-GFP-GM130CΔ10 (J–M) were BFA treated and processed to reveal GFP fluorescence, GRASP65 staining, merged images and, from single optical sections, representations of the colocalized pixels. Bar, 10 µm. GRASP65 recruitment (N) was assayed by determining the fraction of total GFP-positive pixels in single optical sections (chosen to maximize mitochondrial representation) that colocalized with GRASP65 staining (n = 3, ±SEM, >15 cells/experiment; *, P < 0.0001).
Mentions: The absence of clustering by T20-GFP-GM130CΔ10 strongly suggests that T20-GFP-GM130Cterm clusters mitochondria by recruiting endogenous GRASP65. As a test, GRASP65 localization was determined. In BFA-treated cells GRASP65 is known to be principally associated with remnant membrane structures localized adjacent to distributed ER exit sites (Seemann et al., 2000; Ward et al., 2001). Consistent with this localization, GRASP65 was present in dispersed punctate structures in cells expressing T20-GFP, which itself was localized to filamentous mitochondria (Fig. 5, A–D). In striking contrast, GRASP65 localization was largely juxtanuclear in cells expressing T20-GFP-GM130Cterm, and it was clearly evident on the clustered mitochondria (Fig. 5, E–H). In the case of cells expressing T20-GFP-GM130CΔ10, GRASP65 retained the control BFA remnant pattern and was distinct from the filamentous mitochondria (Fig. 5, J–M). GRASP65 coincidence with the GFP constructs was analyzed on a pixel-by-pixel basis and the quantified results indicated significant specific recruitment of endogenous GRASP65 to mitochondria by T20-GFP-GM130Cterm (Fig. 5 N). Thus, the GM130 C terminus recruited endogenous GRASP65 to mitochondria and this induced their clustering. Because GM130 is required for GRASP65 localization to Golgi membranes (Puthenveedu et al., 2006; Kodani and Sutterlin, 2008) and because the GRASP65 binding site in GM130 is required for GM130-dependent Golgi linking (Puthenveedu et al., 2006), these results strongly suggest that GM130 links Golgi ribbons by recruiting GRASP65, which in turn is sufficient to link membranes. Additionally, the results show that endogenous levels of GRASP65 are sufficient to induce mitochondrial clustering, ruling out concerns regarding overexpression of exogenous constructs.

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