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Molecular motors and a spectrin matrix associate with Golgi membranes in vitro.

Fath KR, Trimbur GM, Burgess DR - J. Cell Biol. (1997)

Bottom Line: In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules.Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin.Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
Cytoplasmic dynein is a microtubule minus-end-directed motor that is thought to power the transport of vesicles from the TGN to the apical cortex in polarized epithelial cells. Trans-Golgi enriched membranes, which were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isolated Golgi stacks lack dynein but contain myosin-I (Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. J. Cell Biol. 126:661-675). We show now that Golgi stacks in vitro bind dynein supplied from cytosol in the absence of ATP, and bud small membranes when incubated with cytosol and ATP. Cytosolic dynein binds to regions of stacks that are destined to bud because dynein is present in budded membranes, but absent from stacks after budding. Budded membranes move exclusively towards microtubule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resists extraction by ice-cold Triton X-100. In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.

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Cytoplasmic dynein binds to stacks in an in vitro Golgi  stack budding assay and remains associated specifically with the  budded membranes. (A) Golgi stacks were incubated in various  conditions at 37°C, then pelleted through a 0.5-M sucrose pad at  10,000 g for 15 min (left). The budded membranes that were released from the stacks and remained in the low-speed supernatant were collected by centrifugation through a 0.5 M sucrose  cushion at 259,000 g for 30 min (right). Pelleted stacks and budded membranes were immunoblotted for AP, dynein IC, and myosin-I. Golgi stacks were incubated in the presence or absence of  cytosol and ATP or with the addition of apyrase to hydrolyze  ATP, or ADP in place of ATP as indicated above each lane. (B)  Thin section electron micrograph of Golgi stacks that were incubated with cytosol and ATP to initiate budding. Budded vesicles  (arrow) are visible at the ends of the Golgi stacks. (C) The immunoblots in A suggested that ATP was required for efficient budding, therefore to quantify budding we measured the relative levels of the plasma membrane protein AP in budded membranes in  various conditions. The level of AP present in the budded membranes in each condition as determined by quantitative immunoblotting is expressed relative to the levels in the complete budding mixture (Cytosol + ATP), which was set at 100%. The  omission of ATP, the inclusion of apyrase or ADP, decreased the  level of AP in budded membranes 4–5-fold. (D) Negative stain  electron micrograph showing that budded membrane pellets contained 50–200-nm vesicles. Bars: 100 nm.
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Figure 2: Cytoplasmic dynein binds to stacks in an in vitro Golgi stack budding assay and remains associated specifically with the budded membranes. (A) Golgi stacks were incubated in various conditions at 37°C, then pelleted through a 0.5-M sucrose pad at 10,000 g for 15 min (left). The budded membranes that were released from the stacks and remained in the low-speed supernatant were collected by centrifugation through a 0.5 M sucrose cushion at 259,000 g for 30 min (right). Pelleted stacks and budded membranes were immunoblotted for AP, dynein IC, and myosin-I. Golgi stacks were incubated in the presence or absence of cytosol and ATP or with the addition of apyrase to hydrolyze ATP, or ADP in place of ATP as indicated above each lane. (B) Thin section electron micrograph of Golgi stacks that were incubated with cytosol and ATP to initiate budding. Budded vesicles (arrow) are visible at the ends of the Golgi stacks. (C) The immunoblots in A suggested that ATP was required for efficient budding, therefore to quantify budding we measured the relative levels of the plasma membrane protein AP in budded membranes in various conditions. The level of AP present in the budded membranes in each condition as determined by quantitative immunoblotting is expressed relative to the levels in the complete budding mixture (Cytosol + ATP), which was set at 100%. The omission of ATP, the inclusion of apyrase or ADP, decreased the level of AP in budded membranes 4–5-fold. (D) Negative stain electron micrograph showing that budded membrane pellets contained 50–200-nm vesicles. Bars: 100 nm.

Mentions: To examine further the regulation of dynein binding to membranes, we adapted an in vitro Golgi budding assay that was developed for isolated rat liver Golgi membranes (Salamero et al., 1990). Golgi stacks were incubated in the presence or absence of cytosol and ATP at 37°C in buffers that promote budding. Electron microscopy confirmed the appearance of buds from the ends of stack membranes in this assay (Fig. 2 B). The post-budding stacks were separated from budded membranes and cytosol by pelleting the stacks through a sucrose pad at 10,000 g. The budded membranes, which remained in the supernatant, were subsequently separated from cytosol by pelleting at 259,000 g through sucrose. Negative stain electron microscopy of the budded membrane pellet shows that the budded membranes are comprised of 50–200-nm vesicles (Fig. 2 D) and are approximately the same size as the buds at the ends of Golgi stacks observed in thin sections (Fig. 2 B).


Molecular motors and a spectrin matrix associate with Golgi membranes in vitro.

Fath KR, Trimbur GM, Burgess DR - J. Cell Biol. (1997)

Cytoplasmic dynein binds to stacks in an in vitro Golgi  stack budding assay and remains associated specifically with the  budded membranes. (A) Golgi stacks were incubated in various  conditions at 37°C, then pelleted through a 0.5-M sucrose pad at  10,000 g for 15 min (left). The budded membranes that were released from the stacks and remained in the low-speed supernatant were collected by centrifugation through a 0.5 M sucrose  cushion at 259,000 g for 30 min (right). Pelleted stacks and budded membranes were immunoblotted for AP, dynein IC, and myosin-I. Golgi stacks were incubated in the presence or absence of  cytosol and ATP or with the addition of apyrase to hydrolyze  ATP, or ADP in place of ATP as indicated above each lane. (B)  Thin section electron micrograph of Golgi stacks that were incubated with cytosol and ATP to initiate budding. Budded vesicles  (arrow) are visible at the ends of the Golgi stacks. (C) The immunoblots in A suggested that ATP was required for efficient budding, therefore to quantify budding we measured the relative levels of the plasma membrane protein AP in budded membranes in  various conditions. The level of AP present in the budded membranes in each condition as determined by quantitative immunoblotting is expressed relative to the levels in the complete budding mixture (Cytosol + ATP), which was set at 100%. The  omission of ATP, the inclusion of apyrase or ADP, decreased the  level of AP in budded membranes 4–5-fold. (D) Negative stain  electron micrograph showing that budded membrane pellets contained 50–200-nm vesicles. Bars: 100 nm.
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Figure 2: Cytoplasmic dynein binds to stacks in an in vitro Golgi stack budding assay and remains associated specifically with the budded membranes. (A) Golgi stacks were incubated in various conditions at 37°C, then pelleted through a 0.5-M sucrose pad at 10,000 g for 15 min (left). The budded membranes that were released from the stacks and remained in the low-speed supernatant were collected by centrifugation through a 0.5 M sucrose cushion at 259,000 g for 30 min (right). Pelleted stacks and budded membranes were immunoblotted for AP, dynein IC, and myosin-I. Golgi stacks were incubated in the presence or absence of cytosol and ATP or with the addition of apyrase to hydrolyze ATP, or ADP in place of ATP as indicated above each lane. (B) Thin section electron micrograph of Golgi stacks that were incubated with cytosol and ATP to initiate budding. Budded vesicles (arrow) are visible at the ends of the Golgi stacks. (C) The immunoblots in A suggested that ATP was required for efficient budding, therefore to quantify budding we measured the relative levels of the plasma membrane protein AP in budded membranes in various conditions. The level of AP present in the budded membranes in each condition as determined by quantitative immunoblotting is expressed relative to the levels in the complete budding mixture (Cytosol + ATP), which was set at 100%. The omission of ATP, the inclusion of apyrase or ADP, decreased the level of AP in budded membranes 4–5-fold. (D) Negative stain electron micrograph showing that budded membrane pellets contained 50–200-nm vesicles. Bars: 100 nm.
Mentions: To examine further the regulation of dynein binding to membranes, we adapted an in vitro Golgi budding assay that was developed for isolated rat liver Golgi membranes (Salamero et al., 1990). Golgi stacks were incubated in the presence or absence of cytosol and ATP at 37°C in buffers that promote budding. Electron microscopy confirmed the appearance of buds from the ends of stack membranes in this assay (Fig. 2 B). The post-budding stacks were separated from budded membranes and cytosol by pelleting the stacks through a sucrose pad at 10,000 g. The budded membranes, which remained in the supernatant, were subsequently separated from cytosol by pelleting at 259,000 g through sucrose. Negative stain electron microscopy of the budded membrane pellet shows that the budded membranes are comprised of 50–200-nm vesicles (Fig. 2 D) and are approximately the same size as the buds at the ends of Golgi stacks observed in thin sections (Fig. 2 B).

Bottom Line: In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules.Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin.Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
Cytoplasmic dynein is a microtubule minus-end-directed motor that is thought to power the transport of vesicles from the TGN to the apical cortex in polarized epithelial cells. Trans-Golgi enriched membranes, which were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isolated Golgi stacks lack dynein but contain myosin-I (Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. J. Cell Biol. 126:661-675). We show now that Golgi stacks in vitro bind dynein supplied from cytosol in the absence of ATP, and bud small membranes when incubated with cytosol and ATP. Cytosolic dynein binds to regions of stacks that are destined to bud because dynein is present in budded membranes, but absent from stacks after budding. Budded membranes move exclusively towards microtubule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resists extraction by ice-cold Triton X-100. In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.

Show MeSH
Related in: MedlinePlus