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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 hHK3 localizes to the Golgi complex. The localization of endogenous Hook proteins in Hep2 cells (A, D, G, J, and M) was compared with the Golgi marker FTCD (B, E, and K), microtubules (H), or ERGIC-53 (N). hHK1 localized to discrete unidentified subcellular structures (A) that do not significantly overlap with the Golgi complex (B). hHK2 localized to discrete subcellular structures that were often observed in linear tracks (D and G), which colocalized with microtubules (H and I). Much of hHK3 staining (J) precisely colocalized with a marker of the cis- and/or medial-Golgi (K), FTCD (Bashour and Bloom 1998). hHK3 labeling detected outside the Golgi (M–O, inset) did not colocalize with ERGIC-53, a marker for ER-to-Golgi intermediates (M–O). In the merged images (C, F, I, L, and O), staining for FTCD, microtubules, or ERGIC-53 is shown in green; and the Hook proteins, in red.
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Figure 3: Endogenous hHK3 localizes to the Golgi complex. The localization of endogenous Hook proteins in Hep2 cells (A, D, G, J, and M) was compared with the Golgi marker FTCD (B, E, and K), microtubules (H), or ERGIC-53 (N). hHK1 localized to discrete unidentified subcellular structures (A) that do not significantly overlap with the Golgi complex (B). hHK2 localized to discrete subcellular structures that were often observed in linear tracks (D and G), which colocalized with microtubules (H and I). Much of hHK3 staining (J) precisely colocalized with a marker of the cis- and/or medial-Golgi (K), FTCD (Bashour and Bloom 1998). hHK3 labeling detected outside the Golgi (M–O, inset) did not colocalize with ERGIC-53, a marker for ER-to-Golgi intermediates (M–O). In the merged images (C, F, I, L, and O), staining for FTCD, microtubules, or ERGIC-53 is shown in green; and the Hook proteins, in red.

Mentions: A distinct role for each of the Hook proteins was also supported by their different subcellular localizations. Endogenous Hook proteins were localized using confocal microscopy and double immunofluorescence labeling with well-characterized markers in Hep2 and Vero cells (Fig. 3; and data not shown). hHK1 (Fig. 3 A) and hHK2 (Fig. 3 D) localize to discrete punctate subcellular structures. Especially in the case of hHK2, the structures appeared often closely associated with microtubules. This was most easily visualized in the thin layer of cytosol just above the nucleus (Fig. 3, G–I). The identity of these structures has been elusive thus far: neither hHK1 nor hHK2 colocalized with antibodies labeling early endosomes (anti-EEA1 or antitransferrin receptors), MVBs (6C4), late endosomes (anti-M6PR), lysosomes (Lamp1), the ER (Calnexin), Golgi complexes (FTDC), or mitochondria (Cox1).


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 hHK3 localizes to the Golgi complex. The localization of endogenous Hook proteins in Hep2 cells (A, D, G, J, and M) was compared with the Golgi marker FTCD (B, E, and K), microtubules (H), or ERGIC-53 (N). hHK1 localized to discrete unidentified subcellular structures (A) that do not significantly overlap with the Golgi complex (B). hHK2 localized to discrete subcellular structures that were often observed in linear tracks (D and G), which colocalized with microtubules (H and I). Much of hHK3 staining (J) precisely colocalized with a marker of the cis- and/or medial-Golgi (K), FTCD (Bashour and Bloom 1998). hHK3 labeling detected outside the Golgi (M–O, inset) did not colocalize with ERGIC-53, a marker for ER-to-Golgi intermediates (M–O). In the merged images (C, F, I, L, and O), staining for FTCD, microtubules, or ERGIC-53 is shown in green; and the Hook proteins, in red.
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Related In: Results  -  Collection

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Figure 3: Endogenous hHK3 localizes to the Golgi complex. The localization of endogenous Hook proteins in Hep2 cells (A, D, G, J, and M) was compared with the Golgi marker FTCD (B, E, and K), microtubules (H), or ERGIC-53 (N). hHK1 localized to discrete unidentified subcellular structures (A) that do not significantly overlap with the Golgi complex (B). hHK2 localized to discrete subcellular structures that were often observed in linear tracks (D and G), which colocalized with microtubules (H and I). Much of hHK3 staining (J) precisely colocalized with a marker of the cis- and/or medial-Golgi (K), FTCD (Bashour and Bloom 1998). hHK3 labeling detected outside the Golgi (M–O, inset) did not colocalize with ERGIC-53, a marker for ER-to-Golgi intermediates (M–O). In the merged images (C, F, I, L, and O), staining for FTCD, microtubules, or ERGIC-53 is shown in green; and the Hook proteins, in red.
Mentions: A distinct role for each of the Hook proteins was also supported by their different subcellular localizations. Endogenous Hook proteins were localized using confocal microscopy and double immunofluorescence labeling with well-characterized markers in Hep2 and Vero cells (Fig. 3; and data not shown). hHK1 (Fig. 3 A) and hHK2 (Fig. 3 D) localize to discrete punctate subcellular structures. Especially in the case of hHK2, the structures appeared often closely associated with microtubules. This was most easily visualized in the thin layer of cytosol just above the nucleus (Fig. 3, G–I). The identity of these structures has been elusive thus far: neither hHK1 nor hHK2 colocalized with antibodies labeling early endosomes (anti-EEA1 or antitransferrin receptors), MVBs (6C4), late endosomes (anti-M6PR), lysosomes (Lamp1), the ER (Calnexin), Golgi complexes (FTDC), or mitochondria (Cox1).

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