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A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton.

Gao Y, Sztul E - J. Cell Biol. (2001)

Bottom Line: We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro.Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells.The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.

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FTCD promotes binding of Golgi elements to vimentin. (A) Isolated Golgi elements were incubated either with bacterially expressed and purified FTCD (SG + FTCD + vimentin) or with control bacterial lysate (SG + control lysate + vimentin). The mixtures were loaded at the bottom of a sucrose density gradient and subjected to equilibrium centrifugation to separate soluble proteins from the Golgi membranes. Membrane-containing fractions were combined and supplemented with recombinant vimentin. The mixtures were loaded at the bottom of a sucrose density gradient and subjected to another equilibrium centrifugation. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC, and immunoblotted with antivimentin, anti-GM130, and anti-FTCD antibodies. A proportion of vimentin binds to Golgi elements in the absence of exogenously added FTCD (SG + control lysate + vimentin), but vimentin binding is significantly increased by preincubating the Golgi elements with FTCD (SG + FTCD + vimentin). (B) Poly-l-lysine–coated microtiter wells containing adsorbed vimentin (+) or lacking vimentin (−) received bacterially expressed and purified FTCD, a mixture of FTCD and goat antivimentin antiserum, or a mixture of FTCD and goat nonimmune antiserum. After washing, bound material from triplicate wells was recovered with SDS-PAGE sample buffer and pooled. The pooled material was processed by SDS-PAGE, transferred to NC, and immunoblotted with anti-FTCD antibodies. Vimentin mediates FTCD binding to the wells since FTCD is recovered in wells containing adsorbed vimentin but not in wells lacking vimentin, and FTCD binding is inhibited by antivimentin antibodies. (C) Poly-l-lysine–coated microtiter wells lacking vimentin or containing adsorbed vimentin received buffer, or increasing amounts of bacterially expressed and purified FTCD. Each experimental group contained four wells. After washing to remove unbound FTCD, isolated Golgi elements were added to the wells. After washing to remove unbound Golgi elements, antigiantin antibodies and HRP-conjugated secondary antibodies were added sequentially, followed by an HRP-based colorimetric assay. Background value without Golgi addition has been subtracted from each group. The amount of Golgi binding to vimentin-containing wells without FTCD was set as 1. Each value is an average of three independent experiments, and bars indicate SD. Golgi elements preferentially bind to wells containing adsorbed vimentin, and this association is increased by FTCD in a dose-dependent manner.
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Figure 9: FTCD promotes binding of Golgi elements to vimentin. (A) Isolated Golgi elements were incubated either with bacterially expressed and purified FTCD (SG + FTCD + vimentin) or with control bacterial lysate (SG + control lysate + vimentin). The mixtures were loaded at the bottom of a sucrose density gradient and subjected to equilibrium centrifugation to separate soluble proteins from the Golgi membranes. Membrane-containing fractions were combined and supplemented with recombinant vimentin. The mixtures were loaded at the bottom of a sucrose density gradient and subjected to another equilibrium centrifugation. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC, and immunoblotted with antivimentin, anti-GM130, and anti-FTCD antibodies. A proportion of vimentin binds to Golgi elements in the absence of exogenously added FTCD (SG + control lysate + vimentin), but vimentin binding is significantly increased by preincubating the Golgi elements with FTCD (SG + FTCD + vimentin). (B) Poly-l-lysine–coated microtiter wells containing adsorbed vimentin (+) or lacking vimentin (−) received bacterially expressed and purified FTCD, a mixture of FTCD and goat antivimentin antiserum, or a mixture of FTCD and goat nonimmune antiserum. After washing, bound material from triplicate wells was recovered with SDS-PAGE sample buffer and pooled. The pooled material was processed by SDS-PAGE, transferred to NC, and immunoblotted with anti-FTCD antibodies. Vimentin mediates FTCD binding to the wells since FTCD is recovered in wells containing adsorbed vimentin but not in wells lacking vimentin, and FTCD binding is inhibited by antivimentin antibodies. (C) Poly-l-lysine–coated microtiter wells lacking vimentin or containing adsorbed vimentin received buffer, or increasing amounts of bacterially expressed and purified FTCD. Each experimental group contained four wells. After washing to remove unbound FTCD, isolated Golgi elements were added to the wells. After washing to remove unbound Golgi elements, antigiantin antibodies and HRP-conjugated secondary antibodies were added sequentially, followed by an HRP-based colorimetric assay. Background value without Golgi addition has been subtracted from each group. The amount of Golgi binding to vimentin-containing wells without FTCD was set as 1. Each value is an average of three independent experiments, and bars indicate SD. Golgi elements preferentially bind to wells containing adsorbed vimentin, and this association is increased by FTCD in a dose-dependent manner.

Mentions: To explore the possible linking function of FTCD, we first tested whether FTCD can promote Golgi–vimentin interactions using a density gradient centrifugation assay. Isolated Golgi elements were supplemented either with bacterially expressed and purified FTCD or with control bacterial lysate without FTCD, loaded at the bottom of a sucrose density gradient, and centrifuged. Upfloated membranes from each group were incubated with vimentin and subjected to another round of equilibrium centrifugation. The fractions were collected and tested for their content of vimentin, GM130, and FTCD. As shown in Fig. 9 A, when Golgi elements are not preincubated with FTCD (SG + control lysate + vimentin), vimentin is predominantly recovered in the load at the bottom of the gradient (fractions 5–11), with small amount recovered in lower density fractions containing the bulk of the Golgi marker GM130 (fractions 2 and 3). A significantly different vimentin distribution pattern is obtained when Golgi elements are preincubated with FTCD. As shown in Fig. 9 A, in the presence of exogenous FTCD (SG + FTCD + vimentin), a significantly larger proportion of vimentin is recovered in the Golgi containing fractions. Vimentin is most enriched in fraction 3, which contains the bulk of Golgi elements and the bulk of bound FTCD. These data suggest that addition of FTCD promotes the association between Golgi elements and vimentin.


A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton.

Gao Y, Sztul E - J. Cell Biol. (2001)

FTCD promotes binding of Golgi elements to vimentin. (A) Isolated Golgi elements were incubated either with bacterially expressed and purified FTCD (SG + FTCD + vimentin) or with control bacterial lysate (SG + control lysate + vimentin). The mixtures were loaded at the bottom of a sucrose density gradient and subjected to equilibrium centrifugation to separate soluble proteins from the Golgi membranes. Membrane-containing fractions were combined and supplemented with recombinant vimentin. The mixtures were loaded at the bottom of a sucrose density gradient and subjected to another equilibrium centrifugation. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC, and immunoblotted with antivimentin, anti-GM130, and anti-FTCD antibodies. A proportion of vimentin binds to Golgi elements in the absence of exogenously added FTCD (SG + control lysate + vimentin), but vimentin binding is significantly increased by preincubating the Golgi elements with FTCD (SG + FTCD + vimentin). (B) Poly-l-lysine–coated microtiter wells containing adsorbed vimentin (+) or lacking vimentin (−) received bacterially expressed and purified FTCD, a mixture of FTCD and goat antivimentin antiserum, or a mixture of FTCD and goat nonimmune antiserum. After washing, bound material from triplicate wells was recovered with SDS-PAGE sample buffer and pooled. The pooled material was processed by SDS-PAGE, transferred to NC, and immunoblotted with anti-FTCD antibodies. Vimentin mediates FTCD binding to the wells since FTCD is recovered in wells containing adsorbed vimentin but not in wells lacking vimentin, and FTCD binding is inhibited by antivimentin antibodies. (C) Poly-l-lysine–coated microtiter wells lacking vimentin or containing adsorbed vimentin received buffer, or increasing amounts of bacterially expressed and purified FTCD. Each experimental group contained four wells. After washing to remove unbound FTCD, isolated Golgi elements were added to the wells. After washing to remove unbound Golgi elements, antigiantin antibodies and HRP-conjugated secondary antibodies were added sequentially, followed by an HRP-based colorimetric assay. Background value without Golgi addition has been subtracted from each group. The amount of Golgi binding to vimentin-containing wells without FTCD was set as 1. Each value is an average of three independent experiments, and bars indicate SD. Golgi elements preferentially bind to wells containing adsorbed vimentin, and this association is increased by FTCD in a dose-dependent manner.
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Related In: Results  -  Collection

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Figure 9: FTCD promotes binding of Golgi elements to vimentin. (A) Isolated Golgi elements were incubated either with bacterially expressed and purified FTCD (SG + FTCD + vimentin) or with control bacterial lysate (SG + control lysate + vimentin). The mixtures were loaded at the bottom of a sucrose density gradient and subjected to equilibrium centrifugation to separate soluble proteins from the Golgi membranes. Membrane-containing fractions were combined and supplemented with recombinant vimentin. The mixtures were loaded at the bottom of a sucrose density gradient and subjected to another equilibrium centrifugation. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC, and immunoblotted with antivimentin, anti-GM130, and anti-FTCD antibodies. A proportion of vimentin binds to Golgi elements in the absence of exogenously added FTCD (SG + control lysate + vimentin), but vimentin binding is significantly increased by preincubating the Golgi elements with FTCD (SG + FTCD + vimentin). (B) Poly-l-lysine–coated microtiter wells containing adsorbed vimentin (+) or lacking vimentin (−) received bacterially expressed and purified FTCD, a mixture of FTCD and goat antivimentin antiserum, or a mixture of FTCD and goat nonimmune antiserum. After washing, bound material from triplicate wells was recovered with SDS-PAGE sample buffer and pooled. The pooled material was processed by SDS-PAGE, transferred to NC, and immunoblotted with anti-FTCD antibodies. Vimentin mediates FTCD binding to the wells since FTCD is recovered in wells containing adsorbed vimentin but not in wells lacking vimentin, and FTCD binding is inhibited by antivimentin antibodies. (C) Poly-l-lysine–coated microtiter wells lacking vimentin or containing adsorbed vimentin received buffer, or increasing amounts of bacterially expressed and purified FTCD. Each experimental group contained four wells. After washing to remove unbound FTCD, isolated Golgi elements were added to the wells. After washing to remove unbound Golgi elements, antigiantin antibodies and HRP-conjugated secondary antibodies were added sequentially, followed by an HRP-based colorimetric assay. Background value without Golgi addition has been subtracted from each group. The amount of Golgi binding to vimentin-containing wells without FTCD was set as 1. Each value is an average of three independent experiments, and bars indicate SD. Golgi elements preferentially bind to wells containing adsorbed vimentin, and this association is increased by FTCD in a dose-dependent manner.
Mentions: To explore the possible linking function of FTCD, we first tested whether FTCD can promote Golgi–vimentin interactions using a density gradient centrifugation assay. Isolated Golgi elements were supplemented either with bacterially expressed and purified FTCD or with control bacterial lysate without FTCD, loaded at the bottom of a sucrose density gradient, and centrifuged. Upfloated membranes from each group were incubated with vimentin and subjected to another round of equilibrium centrifugation. The fractions were collected and tested for their content of vimentin, GM130, and FTCD. As shown in Fig. 9 A, when Golgi elements are not preincubated with FTCD (SG + control lysate + vimentin), vimentin is predominantly recovered in the load at the bottom of the gradient (fractions 5–11), with small amount recovered in lower density fractions containing the bulk of the Golgi marker GM130 (fractions 2 and 3). A significantly different vimentin distribution pattern is obtained when Golgi elements are preincubated with FTCD. As shown in Fig. 9 A, in the presence of exogenous FTCD (SG + FTCD + vimentin), a significantly larger proportion of vimentin is recovered in the Golgi containing fractions. Vimentin is most enriched in fraction 3, which contains the bulk of Golgi elements and the bulk of bound FTCD. These data suggest that addition of FTCD promotes the association between Golgi elements and vimentin.

Bottom Line: We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro.Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells.The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.

Show MeSH
Related in: MedlinePlus