Limits...
The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation.

Diao A, Rahman D, Pappin DJ, Lucocq J, Lowe M - J. Cell Biol. (2003)

Bottom Line: Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks.These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus.Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only approximately 25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

Show MeSH

Related in: MedlinePlus

Identification of golgin-84 as a mitotic phosphoprotein. (a–c) Rat liver Golgi membranes were incubated with interphase or mitotic HeLa cytosol in the presence of [γ-32P]ATP for 30 min at 30°C and reisolated by centrifugation. (a) Samples were subjected to 16-BAC/SDS-PAGE 2D electrophoresis and radiolabeled proteins were detected by autoradiography. Positions of the known mitotic phosphoproteins GM130, GRASP65, GRASP55, and rab1 are indicated. (b) The radiolabeled membranes were washed with sodium carbonate and the carbonate pellet was extracted with Triton X-114. Proteins partitioning into the aqueous phase were analyzed by 2D 16-BAC/SDS-PAGE and silver staining, followed by autoradiography. Arrows indicate the doublet of spots corresponding to golgin-84. The lower spot is a proteolytic cleavage product. (c) The radiolabeled membranes were solubilized in SDS and subjected to immunoprecipitation with antibodies to golgin-84. The membrane extracts and immunoprecipitates were analyzed by 1D SDS-PAGE followed by autoradiography. (d) Golgi membranes incubated with interphase or mitotic cytosol (in vitro) or total membrane fractions prepared from interphase and mitotic HeLa cells (in vivo) were analyzed by 1D SDS-PAGE and immunoblotting with antibodies to golgin-84.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172652&req=5

fig1: Identification of golgin-84 as a mitotic phosphoprotein. (a–c) Rat liver Golgi membranes were incubated with interphase or mitotic HeLa cytosol in the presence of [γ-32P]ATP for 30 min at 30°C and reisolated by centrifugation. (a) Samples were subjected to 16-BAC/SDS-PAGE 2D electrophoresis and radiolabeled proteins were detected by autoradiography. Positions of the known mitotic phosphoproteins GM130, GRASP65, GRASP55, and rab1 are indicated. (b) The radiolabeled membranes were washed with sodium carbonate and the carbonate pellet was extracted with Triton X-114. Proteins partitioning into the aqueous phase were analyzed by 2D 16-BAC/SDS-PAGE and silver staining, followed by autoradiography. Arrows indicate the doublet of spots corresponding to golgin-84. The lower spot is a proteolytic cleavage product. (c) The radiolabeled membranes were solubilized in SDS and subjected to immunoprecipitation with antibodies to golgin-84. The membrane extracts and immunoprecipitates were analyzed by 1D SDS-PAGE followed by autoradiography. (d) Golgi membranes incubated with interphase or mitotic cytosol (in vitro) or total membrane fractions prepared from interphase and mitotic HeLa cells (in vivo) were analyzed by 1D SDS-PAGE and immunoblotting with antibodies to golgin-84.

Mentions: To identify mitotic Golgi phosphoproteins, Golgi membranes were incubated with interphase or mitotic cytosol in the presence of [γ-32P]ATP and analyzed by two-dimensional (2D) 16-BAC/SDS-PAGE followed by autoradiography. 20 spots were present exclusively in the mitotic sample (Fig. 1 a). The previously known mitotic phosphoproteins GM130 (Nakamura et al., 1997), GRASP65 (Barr et al., 1997), GRASP55 (Jesch et al., 2001), and rab1 (Bailly et al., 1991) were identified using a combination of mass spectrometry and Western blotting (Fig. 1 a; unpublished data). To identify the other mitotic phosphoproteins, we decided to first fractionate the membranes using sodium carbonate extraction and Triton X-114 phase partitioning in order to simplify the gel pattern before cutting out the spots and analyzing them by mass spectrometry. Analysis of the carbonate pellet/Triton X-114 aqueous phase revealed that only one mitotic phosphoprotein was present in this fraction (Fig. 1 b). This was identified using mass spectrometry as golgin-84, a previously identified coiled-coil protein of unknown function, localized to the Golgi apparatus (Bascom et al., 1999).


The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation.

Diao A, Rahman D, Pappin DJ, Lucocq J, Lowe M - J. Cell Biol. (2003)

Identification of golgin-84 as a mitotic phosphoprotein. (a–c) Rat liver Golgi membranes were incubated with interphase or mitotic HeLa cytosol in the presence of [γ-32P]ATP for 30 min at 30°C and reisolated by centrifugation. (a) Samples were subjected to 16-BAC/SDS-PAGE 2D electrophoresis and radiolabeled proteins were detected by autoradiography. Positions of the known mitotic phosphoproteins GM130, GRASP65, GRASP55, and rab1 are indicated. (b) The radiolabeled membranes were washed with sodium carbonate and the carbonate pellet was extracted with Triton X-114. Proteins partitioning into the aqueous phase were analyzed by 2D 16-BAC/SDS-PAGE and silver staining, followed by autoradiography. Arrows indicate the doublet of spots corresponding to golgin-84. The lower spot is a proteolytic cleavage product. (c) The radiolabeled membranes were solubilized in SDS and subjected to immunoprecipitation with antibodies to golgin-84. The membrane extracts and immunoprecipitates were analyzed by 1D SDS-PAGE followed by autoradiography. (d) Golgi membranes incubated with interphase or mitotic cytosol (in vitro) or total membrane fractions prepared from interphase and mitotic HeLa cells (in vivo) were analyzed by 1D SDS-PAGE and immunoblotting with antibodies to golgin-84.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172652&req=5

fig1: Identification of golgin-84 as a mitotic phosphoprotein. (a–c) Rat liver Golgi membranes were incubated with interphase or mitotic HeLa cytosol in the presence of [γ-32P]ATP for 30 min at 30°C and reisolated by centrifugation. (a) Samples were subjected to 16-BAC/SDS-PAGE 2D electrophoresis and radiolabeled proteins were detected by autoradiography. Positions of the known mitotic phosphoproteins GM130, GRASP65, GRASP55, and rab1 are indicated. (b) The radiolabeled membranes were washed with sodium carbonate and the carbonate pellet was extracted with Triton X-114. Proteins partitioning into the aqueous phase were analyzed by 2D 16-BAC/SDS-PAGE and silver staining, followed by autoradiography. Arrows indicate the doublet of spots corresponding to golgin-84. The lower spot is a proteolytic cleavage product. (c) The radiolabeled membranes were solubilized in SDS and subjected to immunoprecipitation with antibodies to golgin-84. The membrane extracts and immunoprecipitates were analyzed by 1D SDS-PAGE followed by autoradiography. (d) Golgi membranes incubated with interphase or mitotic cytosol (in vitro) or total membrane fractions prepared from interphase and mitotic HeLa cells (in vivo) were analyzed by 1D SDS-PAGE and immunoblotting with antibodies to golgin-84.
Mentions: To identify mitotic Golgi phosphoproteins, Golgi membranes were incubated with interphase or mitotic cytosol in the presence of [γ-32P]ATP and analyzed by two-dimensional (2D) 16-BAC/SDS-PAGE followed by autoradiography. 20 spots were present exclusively in the mitotic sample (Fig. 1 a). The previously known mitotic phosphoproteins GM130 (Nakamura et al., 1997), GRASP65 (Barr et al., 1997), GRASP55 (Jesch et al., 2001), and rab1 (Bailly et al., 1991) were identified using a combination of mass spectrometry and Western blotting (Fig. 1 a; unpublished data). To identify the other mitotic phosphoproteins, we decided to first fractionate the membranes using sodium carbonate extraction and Triton X-114 phase partitioning in order to simplify the gel pattern before cutting out the spots and analyzing them by mass spectrometry. Analysis of the carbonate pellet/Triton X-114 aqueous phase revealed that only one mitotic phosphoprotein was present in this fraction (Fig. 1 b). This was identified using mass spectrometry as golgin-84, a previously identified coiled-coil protein of unknown function, localized to the Golgi apparatus (Bascom et al., 1999).

Bottom Line: Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks.These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus.Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only approximately 25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

Show MeSH
Related in: MedlinePlus