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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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Ankyrin and spectrin are Golgi peripheral membrane  proteins. (A) Isolated Golgi stacks were immunoblotted with  polyclonal antibodies recognizing ankyrin or spectrin to determine whether these proteins are associated with the Golgi apparatus. We found that spectrin was associated with Golgi membranes using antibodies either recognizing α/β-spectrin (Control)  or βIΣ*-spectrin (not shown). Ankyrin was also bound to Golgi  stacks (Control). To examine the mode of association of ankyrin  and spectrin with Golgi membranes, isolated membranes were incubated with PEMS buffer (Control), 0.6 M KI, 0.1 M Na2CO3  pH 11.5, or cold 1% TX-100. The membranes were separated  from extracted proteins by centrifugation through a 0.5 M sucrose pad, then analyzed by immunoblotting. These blots indicate  that both ankyrin and spectrin are tightly bound peripheral membrane proteins that are resistant to extraction with KI and cold  1% TX-100, but are released from the membrane at alkaline pH.  (B)  We also immunoblotted the Golgi membranes for ankyrin  and spectrin following incubation with cytosol. In the presence of  added cytosol, spectrin and ankyrin are present on control and  0.6 M KI-extracted membranes, but are absent from membranes  that were extracted at pH 11.5 or with cold 1% TX-100.
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Figure 8: Ankyrin and spectrin are Golgi peripheral membrane proteins. (A) Isolated Golgi stacks were immunoblotted with polyclonal antibodies recognizing ankyrin or spectrin to determine whether these proteins are associated with the Golgi apparatus. We found that spectrin was associated with Golgi membranes using antibodies either recognizing α/β-spectrin (Control) or βIΣ*-spectrin (not shown). Ankyrin was also bound to Golgi stacks (Control). To examine the mode of association of ankyrin and spectrin with Golgi membranes, isolated membranes were incubated with PEMS buffer (Control), 0.6 M KI, 0.1 M Na2CO3 pH 11.5, or cold 1% TX-100. The membranes were separated from extracted proteins by centrifugation through a 0.5 M sucrose pad, then analyzed by immunoblotting. These blots indicate that both ankyrin and spectrin are tightly bound peripheral membrane proteins that are resistant to extraction with KI and cold 1% TX-100, but are released from the membrane at alkaline pH. (B) We also immunoblotted the Golgi membranes for ankyrin and spectrin following incubation with cytosol. In the presence of added cytosol, spectrin and ankyrin are present on control and 0.6 M KI-extracted membranes, but are absent from membranes that were extracted at pH 11.5 or with cold 1% TX-100.

Mentions: We wished to establish whether the matrix, identified both ultrastructurally and biochemically, that remains associated with the Golgi membrane detergent ghosts, was composed of members of the recently identified Golgi spectrin matrix (Beck et al., 1994; Devarajan et al., 1996). Therefore, we first immunoblotted intact, isolated Golgi stacks and TGN-containing membranes to determine whether spectrin and ankyrin are present on the unextracted membranes. For spectrin, we used either the affinity-purified βspec-1 polyclonal antibody that recognizes βIΣ*-spectrin (Beck et al., 1994) or a polyclonal antibody that was raised against bovine brain fodrin (α/β-spectrin; Burridge et al., 1982). For ankyrin, we immunoblotted with a polyclonal antibody raised against chicken erythrocyte ankyrin. Both spectrin antibodies recognized spectrin in the stack and TGN-containing membrane fractions as an ∼220–230-kD polypeptide(s) (Fig. 8 A). Due to the decreased resolution at high molecular masses on the 10% SDS–polyacrylamide gels used in this study, it is not possible to assign exact molecular masses, nor is it possible to discern whether both α-and β-spectrin are detected by the fodrin antibody. Hereafter, we will call the polypeptide(s) that react with the fodrin antibody α/β-spectrin. In avians, a common 240-kD α-spectrin is found in all spectrin heterodimers (see review by Bement and Mooseker, 1996). The ankyrin antibody recognized an ∼225–240-kD polypeptide in both membrane fractions (Fig. 8 A), which comigrated with a similar-sized polypeptide from chicken RBC membrane ghosts (data not shown). There was also consistently an ∼160-kD ankyrin immunoreactive peptide that may be constitute a second ankyrin isoform (Beck et al., 1997; Devarajan et al., 1996) or an ankyrin proteolytic breakdown product.


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

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

Ankyrin and spectrin are Golgi peripheral membrane  proteins. (A) Isolated Golgi stacks were immunoblotted with  polyclonal antibodies recognizing ankyrin or spectrin to determine whether these proteins are associated with the Golgi apparatus. We found that spectrin was associated with Golgi membranes using antibodies either recognizing α/β-spectrin (Control)  or βIΣ*-spectrin (not shown). Ankyrin was also bound to Golgi  stacks (Control). To examine the mode of association of ankyrin  and spectrin with Golgi membranes, isolated membranes were incubated with PEMS buffer (Control), 0.6 M KI, 0.1 M Na2CO3  pH 11.5, or cold 1% TX-100. The membranes were separated  from extracted proteins by centrifugation through a 0.5 M sucrose pad, then analyzed by immunoblotting. These blots indicate  that both ankyrin and spectrin are tightly bound peripheral membrane proteins that are resistant to extraction with KI and cold  1% TX-100, but are released from the membrane at alkaline pH.  (B)  We also immunoblotted the Golgi membranes for ankyrin  and spectrin following incubation with cytosol. In the presence of  added cytosol, spectrin and ankyrin are present on control and  0.6 M KI-extracted membranes, but are absent from membranes  that were extracted at pH 11.5 or with cold 1% TX-100.
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Figure 8: Ankyrin and spectrin are Golgi peripheral membrane proteins. (A) Isolated Golgi stacks were immunoblotted with polyclonal antibodies recognizing ankyrin or spectrin to determine whether these proteins are associated with the Golgi apparatus. We found that spectrin was associated with Golgi membranes using antibodies either recognizing α/β-spectrin (Control) or βIΣ*-spectrin (not shown). Ankyrin was also bound to Golgi stacks (Control). To examine the mode of association of ankyrin and spectrin with Golgi membranes, isolated membranes were incubated with PEMS buffer (Control), 0.6 M KI, 0.1 M Na2CO3 pH 11.5, or cold 1% TX-100. The membranes were separated from extracted proteins by centrifugation through a 0.5 M sucrose pad, then analyzed by immunoblotting. These blots indicate that both ankyrin and spectrin are tightly bound peripheral membrane proteins that are resistant to extraction with KI and cold 1% TX-100, but are released from the membrane at alkaline pH. (B) We also immunoblotted the Golgi membranes for ankyrin and spectrin following incubation with cytosol. In the presence of added cytosol, spectrin and ankyrin are present on control and 0.6 M KI-extracted membranes, but are absent from membranes that were extracted at pH 11.5 or with cold 1% TX-100.
Mentions: We wished to establish whether the matrix, identified both ultrastructurally and biochemically, that remains associated with the Golgi membrane detergent ghosts, was composed of members of the recently identified Golgi spectrin matrix (Beck et al., 1994; Devarajan et al., 1996). Therefore, we first immunoblotted intact, isolated Golgi stacks and TGN-containing membranes to determine whether spectrin and ankyrin are present on the unextracted membranes. For spectrin, we used either the affinity-purified βspec-1 polyclonal antibody that recognizes βIΣ*-spectrin (Beck et al., 1994) or a polyclonal antibody that was raised against bovine brain fodrin (α/β-spectrin; Burridge et al., 1982). For ankyrin, we immunoblotted with a polyclonal antibody raised against chicken erythrocyte ankyrin. Both spectrin antibodies recognized spectrin in the stack and TGN-containing membrane fractions as an ∼220–230-kD polypeptide(s) (Fig. 8 A). Due to the decreased resolution at high molecular masses on the 10% SDS–polyacrylamide gels used in this study, it is not possible to assign exact molecular masses, nor is it possible to discern whether both α-and β-spectrin are detected by the fodrin antibody. Hereafter, we will call the polypeptide(s) that react with the fodrin antibody α/β-spectrin. In avians, a common 240-kD α-spectrin is found in all spectrin heterodimers (see review by Bement and Mooseker, 1996). The ankyrin antibody recognized an ∼225–240-kD polypeptide in both membrane fractions (Fig. 8 A), which comigrated with a similar-sized polypeptide from chicken RBC membrane ghosts (data not shown). There was also consistently an ∼160-kD ankyrin immunoreactive peptide that may be constitute a second ankyrin isoform (Beck et al., 1997; Devarajan et al., 1996) or an ankyrin proteolytic breakdown product.

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