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The Golgi-associated hook3 protein is a member of a novel family of microtubule-binding proteins.

Walenta JH, Didier AJ, Liu X, Krämer H - J. Cell Biol. (2001)

Bottom Line: Microtubules are central to the spatial organization of diverse membrane-trafficking systems.Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo.Unlike other cis-Golgi-associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization.

View Article: PubMed Central - PubMed

Affiliation: Center for Basic Neuroscience and Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

ABSTRACT
Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH(2)-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi-associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.

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The dHKs bind microtubules. (A) Cytosol from Drosophila tissue culture cells was used in microtubule spin-down assays to test the ability of dHK to associate with bovine microtubules. dHK was found in the pellet associating with microtubules (lane 4) only when Drosophila cytosol and taxol-stabilized microtubules were combined. (B) dHK protein also bound to endogenous Drosophila microtubules when they were stabilized by the addition of GTP–taxol. Tubulin and dHK were pelleted in the presence of taxol–GTP (lane 4) but not in their absence (lane 2).
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Figure 8: The dHKs bind microtubules. (A) Cytosol from Drosophila tissue culture cells was used in microtubule spin-down assays to test the ability of dHK to associate with bovine microtubules. dHK was found in the pellet associating with microtubules (lane 4) only when Drosophila cytosol and taxol-stabilized microtubules were combined. (B) dHK protein also bound to endogenous Drosophila microtubules when they were stabilized by the addition of GTP–taxol. Tubulin and dHK were pelleted in the presence of taxol–GTP (lane 4) but not in their absence (lane 2).

Mentions: Because this NH2-terminal domain exhibits the highest similarity to the dHK (Fig. 1 A), we tested the ability of dHK to bind to microtubules. Standard microtubule spin-down assays demonstrated that dHK from Drosophila cytosol bound exogenous bovine microtubules (Fig. 8 A). To test whether dHK can also bind to Drosophila microtubules, GTP and taxol were used to stabilize endogenous microtubules in Drosophila cytosol. When microtubules were sedimented by high speed centrifugation, dHK was found in the microtubule pellet (Fig. 8 B, lane 4). In the absence of stabilized microtubules, all of the dHK protein remained in the supernatant (Fig. 8 B, lanes 1 and 2). These results indicated that Hook proteins constitute a novel family of microtubule-binding proteins.


The Golgi-associated hook3 protein is a member of a novel family of microtubule-binding proteins.

Walenta JH, Didier AJ, Liu X, Krämer H - J. Cell Biol. (2001)

The dHKs bind microtubules. (A) Cytosol from Drosophila tissue culture cells was used in microtubule spin-down assays to test the ability of dHK to associate with bovine microtubules. dHK was found in the pellet associating with microtubules (lane 4) only when Drosophila cytosol and taxol-stabilized microtubules were combined. (B) dHK protein also bound to endogenous Drosophila microtubules when they were stabilized by the addition of GTP–taxol. Tubulin and dHK were pelleted in the presence of taxol–GTP (lane 4) but not in their absence (lane 2).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198811&req=5

Figure 8: The dHKs bind microtubules. (A) Cytosol from Drosophila tissue culture cells was used in microtubule spin-down assays to test the ability of dHK to associate with bovine microtubules. dHK was found in the pellet associating with microtubules (lane 4) only when Drosophila cytosol and taxol-stabilized microtubules were combined. (B) dHK protein also bound to endogenous Drosophila microtubules when they were stabilized by the addition of GTP–taxol. Tubulin and dHK were pelleted in the presence of taxol–GTP (lane 4) but not in their absence (lane 2).
Mentions: Because this NH2-terminal domain exhibits the highest similarity to the dHK (Fig. 1 A), we tested the ability of dHK to bind to microtubules. Standard microtubule spin-down assays demonstrated that dHK from Drosophila cytosol bound exogenous bovine microtubules (Fig. 8 A). To test whether dHK can also bind to Drosophila microtubules, GTP and taxol were used to stabilize endogenous microtubules in Drosophila cytosol. When microtubules were sedimented by high speed centrifugation, dHK was found in the microtubule pellet (Fig. 8 B, lane 4). In the absence of stabilized microtubules, all of the dHK protein remained in the supernatant (Fig. 8 B, lanes 1 and 2). These results indicated that Hook proteins constitute a novel family of microtubule-binding proteins.

Bottom Line: Microtubules are central to the spatial organization of diverse membrane-trafficking systems.Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo.Unlike other cis-Golgi-associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization.

View Article: PubMed Central - PubMed

Affiliation: Center for Basic Neuroscience and Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

ABSTRACT
Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH(2)-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi-associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.

Show MeSH
Related in: MedlinePlus