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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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Determination of the domain of Hrs required for the inhibition of early endosome fusion. (A) The linear structure of Hrs highlighting some protein motifs. (B) The NH2-terminal half of Hrs, including the VHS and FYVE domains, had no effect on early endosome fusion. (C) Although both helical domains of Hrs inhibit early endosome fusion with similar efficacy to the full-length protein, the minimal fragment of Hrs that is both necessary and sufficient for the recapitulation of the inhibition of early endosome fusion observed with full-length Hrs is the Q-SNARE domain Hrs(515–562).
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fig5: Determination of the domain of Hrs required for the inhibition of early endosome fusion. (A) The linear structure of Hrs highlighting some protein motifs. (B) The NH2-terminal half of Hrs, including the VHS and FYVE domains, had no effect on early endosome fusion. (C) Although both helical domains of Hrs inhibit early endosome fusion with similar efficacy to the full-length protein, the minimal fragment of Hrs that is both necessary and sufficient for the recapitulation of the inhibition of early endosome fusion observed with full-length Hrs is the Q-SNARE domain Hrs(515–562).

Mentions: To understand the mechanism by which Hrs inhibited early endosome fusion, we examined the effect of different domains of Hrs to determine whether a minimal fragment of Hrs was required for the effect. We examined a large NH2-terminal fragment of Hrs that contains the VHS, FYVE, and UIM domains, as well as the binding sites for eps15 (Bean et al., 2000) and STAM (Asao et al., 1997; Fig. 5Figure 5.


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)

Determination of the domain of Hrs required for the inhibition of early endosome fusion. (A) The linear structure of Hrs highlighting some protein motifs. (B) The NH2-terminal half of Hrs, including the VHS and FYVE domains, had no effect on early endosome fusion. (C) Although both helical domains of Hrs inhibit early endosome fusion with similar efficacy to the full-length protein, the minimal fragment of Hrs that is both necessary and sufficient for the recapitulation of the inhibition of early endosome fusion observed with full-length Hrs is the Q-SNARE domain Hrs(515–562).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Determination of the domain of Hrs required for the inhibition of early endosome fusion. (A) The linear structure of Hrs highlighting some protein motifs. (B) The NH2-terminal half of Hrs, including the VHS and FYVE domains, had no effect on early endosome fusion. (C) Although both helical domains of Hrs inhibit early endosome fusion with similar efficacy to the full-length protein, the minimal fragment of Hrs that is both necessary and sufficient for the recapitulation of the inhibition of early endosome fusion observed with full-length Hrs is the Q-SNARE domain Hrs(515–562).
Mentions: To understand the mechanism by which Hrs inhibited early endosome fusion, we examined the effect of different domains of Hrs to determine whether a minimal fragment of Hrs was required for the effect. We examined a large NH2-terminal fragment of Hrs that contains the VHS, FYVE, and UIM domains, as well as the binding sites for eps15 (Bean et al., 2000) and STAM (Asao et al., 1997; Fig. 5Figure 5.

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