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Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex.

Sun W, Yan Q, Vida TA, Bean AJ - J. Cell Biol. (2003)

Bottom Line: We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes.SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain.Hrs inhibited formation of the syntaxin 13-SNAP-25-VAMP2 complex by displacing VAMP2 from the complex.

View Article: PubMed Central - PubMed

Affiliation: The University of Texas Health Science Center, Dept. of Neurobiology and Anatomy, 6431 Fannin Street, MSB 7.208, Houston, TX 77030, USA.

ABSTRACT
Movement through the endocytic pathway occurs principally via a series of membrane fusion and fission reactions that allow sorting of molecules to be recycled from those to be degraded. Endosome fusion is dependent on SNARE proteins, although the nature of the proteins involved and their regulation has not been fully elucidated. We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes. A region of Hrs predicted to form a coiled coil required for binding the Q-SNARE, SNAP-25, mimicked the inhibition of endosome fusion produced by full-length Hrs, and was sufficient for endosome binding. SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain. Syntaxin 13 inhibited early endosomal fusion and botulinum toxin/E inhibition of early endosomal fusion was reversed by addition of SNAP-25(150-206), confirming a role for syntaxin 13, and establishing a role for SNAP-25 in endosomal fusion. Hrs inhibited formation of the syntaxin 13-SNAP-25-VAMP2 complex by displacing VAMP2 from the complex. These data suggest that SNAP-25 is a receptor for Hrs on early endosomal membranes and that the binding of Hrs to SNAP-25 on endosomal membranes inhibits formation of a SNARE complex required for homotypic endosome fusion.

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Hrs decreases the efficiency of early endosomal syntaxin13–SNAP-25–VAMP2 complex formation. Quantitation of the amount of Hrs (A, expressed in optical density units) or VAMP (B, expressed as a percentage of VAMP incorporated in the absence of Hrs) incorporated into the 7 S complex in the presence of increasing amounts of Hrs is shown. (C) Glutathione-immobilized GST or GST–syntaxin 13, SNAP-25, and VAMP2 were incubated in the absence (lane 1) and presence of increasing amounts of Hrs. Samples were processed, and immunoblots were visualized with 125I-secondary antibodies and phosphorimaging.
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fig9: Hrs decreases the efficiency of early endosomal syntaxin13–SNAP-25–VAMP2 complex formation. Quantitation of the amount of Hrs (A, expressed in optical density units) or VAMP (B, expressed as a percentage of VAMP incorporated in the absence of Hrs) incorporated into the 7 S complex in the presence of increasing amounts of Hrs is shown. (C) Glutathione-immobilized GST or GST–syntaxin 13, SNAP-25, and VAMP2 were incubated in the absence (lane 1) and presence of increasing amounts of Hrs. Samples were processed, and immunoblots were visualized with 125I-secondary antibodies and phosphorimaging.

Mentions: To understand how Hrs might inhibit early endosome fusion, we examined the in vitro formation of an early endosomal 7S fusion complex containing syntaxin 13, SNAP-25, and VAMP2. Using immobilized syntaxin 13, we formed the 7S complex with SNAP-25 and VAMP2 (Fig. 9Figure 9.


Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex.

Sun W, Yan Q, Vida TA, Bean AJ - J. Cell Biol. (2003)

Hrs decreases the efficiency of early endosomal syntaxin13–SNAP-25–VAMP2 complex formation. Quantitation of the amount of Hrs (A, expressed in optical density units) or VAMP (B, expressed as a percentage of VAMP incorporated in the absence of Hrs) incorporated into the 7 S complex in the presence of increasing amounts of Hrs is shown. (C) Glutathione-immobilized GST or GST–syntaxin 13, SNAP-25, and VAMP2 were incubated in the absence (lane 1) and presence of increasing amounts of Hrs. Samples were processed, and immunoblots were visualized with 125I-secondary antibodies and phosphorimaging.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172712&req=5

fig9: Hrs decreases the efficiency of early endosomal syntaxin13–SNAP-25–VAMP2 complex formation. Quantitation of the amount of Hrs (A, expressed in optical density units) or VAMP (B, expressed as a percentage of VAMP incorporated in the absence of Hrs) incorporated into the 7 S complex in the presence of increasing amounts of Hrs is shown. (C) Glutathione-immobilized GST or GST–syntaxin 13, SNAP-25, and VAMP2 were incubated in the absence (lane 1) and presence of increasing amounts of Hrs. Samples were processed, and immunoblots were visualized with 125I-secondary antibodies and phosphorimaging.
Mentions: To understand how Hrs might inhibit early endosome fusion, we examined the in vitro formation of an early endosomal 7S fusion complex containing syntaxin 13, SNAP-25, and VAMP2. Using immobilized syntaxin 13, we formed the 7S complex with SNAP-25 and VAMP2 (Fig. 9Figure 9.

Bottom Line: We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes.SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain.Hrs inhibited formation of the syntaxin 13-SNAP-25-VAMP2 complex by displacing VAMP2 from the complex.

View Article: PubMed Central - PubMed

Affiliation: The University of Texas Health Science Center, Dept. of Neurobiology and Anatomy, 6431 Fannin Street, MSB 7.208, Houston, TX 77030, USA.

ABSTRACT
Movement through the endocytic pathway occurs principally via a series of membrane fusion and fission reactions that allow sorting of molecules to be recycled from those to be degraded. Endosome fusion is dependent on SNARE proteins, although the nature of the proteins involved and their regulation has not been fully elucidated. We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes. A region of Hrs predicted to form a coiled coil required for binding the Q-SNARE, SNAP-25, mimicked the inhibition of endosome fusion produced by full-length Hrs, and was sufficient for endosome binding. SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain. Syntaxin 13 inhibited early endosomal fusion and botulinum toxin/E inhibition of early endosomal fusion was reversed by addition of SNAP-25(150-206), confirming a role for syntaxin 13, and establishing a role for SNAP-25 in endosomal fusion. Hrs inhibited formation of the syntaxin 13-SNAP-25-VAMP2 complex by displacing VAMP2 from the complex. These data suggest that SNAP-25 is a receptor for Hrs on early endosomal membranes and that the binding of Hrs to SNAP-25 on endosomal membranes inhibits formation of a SNARE complex required for homotypic endosome fusion.

Show MeSH
Related in: MedlinePlus