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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.

Show MeSH
Affinity chromatography of brain membranes using immobilized Hrs(449–562). A SNARE complex consisting of syntaxin 13, SNAP-25, and VAMP2 has been previously reported to be present on early endosomal membranes (Prekeris et al., 1998). Hrs has been previously reported to bind to SNAP-25 (Bean et al., 1997) requiring aa 515–562 (Tsujimoto and Bean, 2000). Candidate proteins were examined by Western analysis on blots obtained from the affinity column eluate. SNAP-25, syntaxin 13, and VAMP2 bound to the column, whereas SV2, eps15, synaptophysin, synaptotagmin, synapsin, syntaxin 6, and rab 5, rab 15, and EEA-1 (not depicted) did not bind to Hrs (449–562).
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fig7: Affinity chromatography of brain membranes using immobilized Hrs(449–562). A SNARE complex consisting of syntaxin 13, SNAP-25, and VAMP2 has been previously reported to be present on early endosomal membranes (Prekeris et al., 1998). Hrs has been previously reported to bind to SNAP-25 (Bean et al., 1997) requiring aa 515–562 (Tsujimoto and Bean, 2000). Candidate proteins were examined by Western analysis on blots obtained from the affinity column eluate. SNAP-25, syntaxin 13, and VAMP2 bound to the column, whereas SV2, eps15, synaptophysin, synaptotagmin, synapsin, syntaxin 6, and rab 5, rab 15, and EEA-1 (not depicted) did not bind to Hrs (449–562).

Mentions: Because Hrs bound saturably to endosomal membranes and this binding was inhibited by SNAP-25(150–206), we hypothesized the presence of a membrane receptor whose identity was likely SNAP-25. To identify potential membrane receptors, affinity chromatography was performed using immobilized Hrs449–562 on a detergent-extracted rat brain membrane fraction. We detected SNAP-25, syntaxin13, and VAMP2 after salt elution from the affinity column, whereas none of these proteins were detected in the eluate from a control (GST) column (Fig. 7)Figure 7.


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)

Affinity chromatography of brain membranes using immobilized Hrs(449–562). A SNARE complex consisting of syntaxin 13, SNAP-25, and VAMP2 has been previously reported to be present on early endosomal membranes (Prekeris et al., 1998). Hrs has been previously reported to bind to SNAP-25 (Bean et al., 1997) requiring aa 515–562 (Tsujimoto and Bean, 2000). Candidate proteins were examined by Western analysis on blots obtained from the affinity column eluate. SNAP-25, syntaxin 13, and VAMP2 bound to the column, whereas SV2, eps15, synaptophysin, synaptotagmin, synapsin, syntaxin 6, and rab 5, rab 15, and EEA-1 (not depicted) did not bind to Hrs (449–562).
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Affinity chromatography of brain membranes using immobilized Hrs(449–562). A SNARE complex consisting of syntaxin 13, SNAP-25, and VAMP2 has been previously reported to be present on early endosomal membranes (Prekeris et al., 1998). Hrs has been previously reported to bind to SNAP-25 (Bean et al., 1997) requiring aa 515–562 (Tsujimoto and Bean, 2000). Candidate proteins were examined by Western analysis on blots obtained from the affinity column eluate. SNAP-25, syntaxin 13, and VAMP2 bound to the column, whereas SV2, eps15, synaptophysin, synaptotagmin, synapsin, syntaxin 6, and rab 5, rab 15, and EEA-1 (not depicted) did not bind to Hrs (449–562).
Mentions: Because Hrs bound saturably to endosomal membranes and this binding was inhibited by SNAP-25(150–206), we hypothesized the presence of a membrane receptor whose identity was likely SNAP-25. To identify potential membrane receptors, affinity chromatography was performed using immobilized Hrs449–562 on a detergent-extracted rat brain membrane fraction. We detected SNAP-25, syntaxin13, and VAMP2 after salt elution from the affinity column, whereas none of these proteins were detected in the eluate from a control (GST) column (Fig. 7)Figure 7.

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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