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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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Endogenous human Hook proteins function in distinct complexes. HEK293 postnuclear supernatants were subjected to immunoprecipitation using the indicated hHK antisera (IP αhHK), either directly (−) or after cross-linking with 100 μM BS3 (+). Precipitated Hook proteins were detected with the respective hHK antiserum by Western blotting. Each hHK antiserum is able to specifically immunoprecipitate its respective endogenous protein from noncross-linked samples (hHK1, lane 1; hHK2, lane 9; hHK3, lane 17). In each of the cross-linked samples, high molecular weight complexes are detected with the specific anti-hHK antibodies used for their immunoprecipitation (hHK1, lane 2; hHK2, lane 10; hHK3, lane 18) but not the other two anti-hHK antibodies (lanes 4, 6, 8, 12, 14, and 16). The identity of the bands at ∼85 kD in lanes 3 and 5 has not been determined.
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Figure 2: Endogenous human Hook proteins function in distinct complexes. HEK293 postnuclear supernatants were subjected to immunoprecipitation using the indicated hHK antisera (IP αhHK), either directly (−) or after cross-linking with 100 μM BS3 (+). Precipitated Hook proteins were detected with the respective hHK antiserum by Western blotting. Each hHK antiserum is able to specifically immunoprecipitate its respective endogenous protein from noncross-linked samples (hHK1, lane 1; hHK2, lane 9; hHK3, lane 17). In each of the cross-linked samples, high molecular weight complexes are detected with the specific anti-hHK antibodies used for their immunoprecipitation (hHK1, lane 2; hHK2, lane 10; hHK3, lane 18) but not the other two anti-hHK antibodies (lanes 4, 6, 8, 12, 14, and 16). The identity of the bands at ∼85 kD in lanes 3 and 5 has not been determined.

Mentions: Because of the similarity of coiled coil domains between the three human Hook proteins, we used a combination of cross-linking and immunoprecipitation experiments to determine whether Hook proteins form heterodimers. Postnuclear supernatants from HEK293 cells were immunoprecipitated with antibodies specific for each Hook protein, either directly or after cross-linking with 100 μM BS3. Western analysis of the immunoprecipitates revealed that the endogenous Hook proteins exist as members of three separate protein complexes (Fig. 2). All three human Hook proteins were predominantly found in complexes larger than expected for Hook dimers, but putative additional components of these complexes have not yet been identified. When complexes immunoprecipitated with one antibody were probed for the presence of the two other human Hook proteins, none was detected (Fig. 2, lanes 4, 6, 8, 12, 14, and 16). These results demonstrate that endogenous Hook proteins do not heterodimerize and suggest separate functions for the three human Hooks.


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)

Endogenous human Hook proteins function in distinct complexes. HEK293 postnuclear supernatants were subjected to immunoprecipitation using the indicated hHK antisera (IP αhHK), either directly (−) or after cross-linking with 100 μM BS3 (+). Precipitated Hook proteins were detected with the respective hHK antiserum by Western blotting. Each hHK antiserum is able to specifically immunoprecipitate its respective endogenous protein from noncross-linked samples (hHK1, lane 1; hHK2, lane 9; hHK3, lane 17). In each of the cross-linked samples, high molecular weight complexes are detected with the specific anti-hHK antibodies used for their immunoprecipitation (hHK1, lane 2; hHK2, lane 10; hHK3, lane 18) but not the other two anti-hHK antibodies (lanes 4, 6, 8, 12, 14, and 16). The identity of the bands at ∼85 kD in lanes 3 and 5 has not been determined.
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Related In: Results  -  Collection

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Figure 2: Endogenous human Hook proteins function in distinct complexes. HEK293 postnuclear supernatants were subjected to immunoprecipitation using the indicated hHK antisera (IP αhHK), either directly (−) or after cross-linking with 100 μM BS3 (+). Precipitated Hook proteins were detected with the respective hHK antiserum by Western blotting. Each hHK antiserum is able to specifically immunoprecipitate its respective endogenous protein from noncross-linked samples (hHK1, lane 1; hHK2, lane 9; hHK3, lane 17). In each of the cross-linked samples, high molecular weight complexes are detected with the specific anti-hHK antibodies used for their immunoprecipitation (hHK1, lane 2; hHK2, lane 10; hHK3, lane 18) but not the other two anti-hHK antibodies (lanes 4, 6, 8, 12, 14, and 16). The identity of the bands at ∼85 kD in lanes 3 and 5 has not been determined.
Mentions: Because of the similarity of coiled coil domains between the three human Hook proteins, we used a combination of cross-linking and immunoprecipitation experiments to determine whether Hook proteins form heterodimers. Postnuclear supernatants from HEK293 cells were immunoprecipitated with antibodies specific for each Hook protein, either directly or after cross-linking with 100 μM BS3. Western analysis of the immunoprecipitates revealed that the endogenous Hook proteins exist as members of three separate protein complexes (Fig. 2). All three human Hook proteins were predominantly found in complexes larger than expected for Hook dimers, but putative additional components of these complexes have not yet been identified. When complexes immunoprecipitated with one antibody were probed for the presence of the two other human Hook proteins, none was detected (Fig. 2, lanes 4, 6, 8, 12, 14, and 16). These results demonstrate that endogenous Hook proteins do not heterodimerize and suggest separate functions for the three human Hooks.

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