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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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Mitochondrial clustering by GRASP65. Schematic diagram of the constructs (A). Untransfected HeLa cells (B–E) or cells expressing GFP-ActA (F–I) or G65-GFP-ActA (J–M) were analyzed 24 h after transfection using Mitotracker (red) to stain mitochondria, GFP fluorescence (green) to localize the transfected proteins, and giantin (blue) staining to image the Golgi apparatus. An identical analysis was performed after a 30-min BFA treatment on cells expressing GFP-ActA (N–Q) or G65-GFP-ActA (R–U). Bar, 10 µm. Radial profile plots show the spread of mitochondrial fluorescence starting from the centroid and extending to the cell periphery for cells expressing GFP-ActA (V), G65-GFP-ActA (W), or BFA-treated cells expressing G65-GFP-ActA (X). Values are averages corresponding to the fraction of total fluorescence present in each concentric circle drawn from the centroid (n = 3, ±SEM, >15 cells/experiment).
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fig1: Mitochondrial clustering by GRASP65. Schematic diagram of the constructs (A). Untransfected HeLa cells (B–E) or cells expressing GFP-ActA (F–I) or G65-GFP-ActA (J–M) were analyzed 24 h after transfection using Mitotracker (red) to stain mitochondria, GFP fluorescence (green) to localize the transfected proteins, and giantin (blue) staining to image the Golgi apparatus. An identical analysis was performed after a 30-min BFA treatment on cells expressing GFP-ActA (N–Q) or G65-GFP-ActA (R–U). Bar, 10 µm. Radial profile plots show the spread of mitochondrial fluorescence starting from the centroid and extending to the cell periphery for cells expressing GFP-ActA (V), G65-GFP-ActA (W), or BFA-treated cells expressing G65-GFP-ActA (X). Values are averages corresponding to the fraction of total fluorescence present in each concentric circle drawn from the centroid (n = 3, ±SEM, >15 cells/experiment).

Mentions: As a test of the hypothesis that GRASP65 directly cross-bridges membranes, we expressed in HeLa cells a modified version of GRASP65 containing a C-terminal membrane anchor sequence specifying targeting to the mitochondrial outer membrane. The mitochondrial-targeting signal, derived from the ActA protein of Listeria monocytogenes (Pistor et al., 1994), was placed after an inserted GFP coding sequence at the C terminus of GRASP65 to yield a cytoplasmically disposed G65-GFP-ActA construct (Fig. 1 A). GFP-ActA, which lacked the GRASP65 sequence but was otherwise identical, served as a control.


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)

Mitochondrial clustering by GRASP65. Schematic diagram of the constructs (A). Untransfected HeLa cells (B–E) or cells expressing GFP-ActA (F–I) or G65-GFP-ActA (J–M) were analyzed 24 h after transfection using Mitotracker (red) to stain mitochondria, GFP fluorescence (green) to localize the transfected proteins, and giantin (blue) staining to image the Golgi apparatus. An identical analysis was performed after a 30-min BFA treatment on cells expressing GFP-ActA (N–Q) or G65-GFP-ActA (R–U). Bar, 10 µm. Radial profile plots show the spread of mitochondrial fluorescence starting from the centroid and extending to the cell periphery for cells expressing GFP-ActA (V), G65-GFP-ActA (W), or BFA-treated cells expressing G65-GFP-ActA (X). Values are averages corresponding to the fraction of total fluorescence present in each concentric circle drawn from the centroid (n = 3, ±SEM, >15 cells/experiment).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Mitochondrial clustering by GRASP65. Schematic diagram of the constructs (A). Untransfected HeLa cells (B–E) or cells expressing GFP-ActA (F–I) or G65-GFP-ActA (J–M) were analyzed 24 h after transfection using Mitotracker (red) to stain mitochondria, GFP fluorescence (green) to localize the transfected proteins, and giantin (blue) staining to image the Golgi apparatus. An identical analysis was performed after a 30-min BFA treatment on cells expressing GFP-ActA (N–Q) or G65-GFP-ActA (R–U). Bar, 10 µm. Radial profile plots show the spread of mitochondrial fluorescence starting from the centroid and extending to the cell periphery for cells expressing GFP-ActA (V), G65-GFP-ActA (W), or BFA-treated cells expressing G65-GFP-ActA (X). Values are averages corresponding to the fraction of total fluorescence present in each concentric circle drawn from the centroid (n = 3, ±SEM, >15 cells/experiment).
Mentions: As a test of the hypothesis that GRASP65 directly cross-bridges membranes, we expressed in HeLa cells a modified version of GRASP65 containing a C-terminal membrane anchor sequence specifying targeting to the mitochondrial outer membrane. The mitochondrial-targeting signal, derived from the ActA protein of Listeria monocytogenes (Pistor et al., 1994), was placed after an inserted GFP coding sequence at the C terminus of GRASP65 to yield a cytoplasmically disposed G65-GFP-ActA construct (Fig. 1 A). GFP-ActA, which lacked the GRASP65 sequence but was otherwise identical, served as a control.

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