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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 binds saturably to early endosomal membranes. Hrs and Hrs(449–562) bind to early endosomal membranes. Early endosomes were purified on a discontinuous sucrose gradient and contain the marker protein EEA-1. Full-length Hrs binds saturably to early endosomal membranes (A). SNAP-25(150–206) inhibited the binding of Hrs to endosomal membranes (B). Hrs(449–562) also binds to endosomal membranes (C), suggesting that the coiled-coil domain of Hrs is sufficient for endosomal membrane binding. Hrs or Hrs(449–562) is not found in the pellet in the absence of membranes.
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fig6: Hrs binds saturably to early endosomal membranes. Hrs and Hrs(449–562) bind to early endosomal membranes. Early endosomes were purified on a discontinuous sucrose gradient and contain the marker protein EEA-1. Full-length Hrs binds saturably to early endosomal membranes (A). SNAP-25(150–206) inhibited the binding of Hrs to endosomal membranes (B). Hrs(449–562) also binds to endosomal membranes (C), suggesting that the coiled-coil domain of Hrs is sufficient for endosomal membrane binding. Hrs or Hrs(449–562) is not found in the pellet in the absence of membranes.

Mentions: To determine whether Hrs might exert its effect by binding to endosomal membranes, we incubated purified endosomes with increasing concentrations of recombinant Hrs (Fig. 6Figure 6.


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 binds saturably to early endosomal membranes. Hrs and Hrs(449–562) bind to early endosomal membranes. Early endosomes were purified on a discontinuous sucrose gradient and contain the marker protein EEA-1. Full-length Hrs binds saturably to early endosomal membranes (A). SNAP-25(150–206) inhibited the binding of Hrs to endosomal membranes (B). Hrs(449–562) also binds to endosomal membranes (C), suggesting that the coiled-coil domain of Hrs is sufficient for endosomal membrane binding. Hrs or Hrs(449–562) is not found in the pellet in the absence of membranes.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Hrs binds saturably to early endosomal membranes. Hrs and Hrs(449–562) bind to early endosomal membranes. Early endosomes were purified on a discontinuous sucrose gradient and contain the marker protein EEA-1. Full-length Hrs binds saturably to early endosomal membranes (A). SNAP-25(150–206) inhibited the binding of Hrs to endosomal membranes (B). Hrs(449–562) also binds to endosomal membranes (C), suggesting that the coiled-coil domain of Hrs is sufficient for endosomal membrane binding. Hrs or Hrs(449–562) is not found in the pellet in the absence of membranes.
Mentions: To determine whether Hrs might exert its effect by binding to endosomal membranes, we incubated purified endosomes with increasing concentrations of recombinant Hrs (Fig. 6Figure 6.

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