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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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Morphology of endosomal membranes before and after fusion reactions. Membranes were obtained as described for homotypic reactions of early endosomes (see Materials and methods) and fixed in 3% glutaraldehyde (A, donor) or after incubation with acceptor membranes, cytosol, and ATP regenerating system (B, after fusion). The diameter of all membrane bound profiles was measured on images from donor and fused samples (C). The mean diameter of donor compartments was 58.3 ± 1.7 nm (n = 123) and postfusion compartment diameter was 188.5 ± 7.3 nm (n = 96; *, P ≤ 0.0001). The error bars show SEM. Bars: (A and B) 100 nm.
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fig3: Morphology of endosomal membranes before and after fusion reactions. Membranes were obtained as described for homotypic reactions of early endosomes (see Materials and methods) and fixed in 3% glutaraldehyde (A, donor) or after incubation with acceptor membranes, cytosol, and ATP regenerating system (B, after fusion). The diameter of all membrane bound profiles was measured on images from donor and fused samples (C). The mean diameter of donor compartments was 58.3 ± 1.7 nm (n = 123) and postfusion compartment diameter was 188.5 ± 7.3 nm (n = 96; *, P ≤ 0.0001). The error bars show SEM. Bars: (A and B) 100 nm.

Mentions: Ultrastructural examination of the morphology of donor/acceptor membranes before a fusion reaction revealed the presence of consistently sized membrane-bound compartments (mean diameter, 58.3 ± 1.7 nm; Fig. 3Figure 3.


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)

Morphology of endosomal membranes before and after fusion reactions. Membranes were obtained as described for homotypic reactions of early endosomes (see Materials and methods) and fixed in 3% glutaraldehyde (A, donor) or after incubation with acceptor membranes, cytosol, and ATP regenerating system (B, after fusion). The diameter of all membrane bound profiles was measured on images from donor and fused samples (C). The mean diameter of donor compartments was 58.3 ± 1.7 nm (n = 123) and postfusion compartment diameter was 188.5 ± 7.3 nm (n = 96; *, P ≤ 0.0001). The error bars show SEM. Bars: (A and B) 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172712&req=5

fig3: Morphology of endosomal membranes before and after fusion reactions. Membranes were obtained as described for homotypic reactions of early endosomes (see Materials and methods) and fixed in 3% glutaraldehyde (A, donor) or after incubation with acceptor membranes, cytosol, and ATP regenerating system (B, after fusion). The diameter of all membrane bound profiles was measured on images from donor and fused samples (C). The mean diameter of donor compartments was 58.3 ± 1.7 nm (n = 123) and postfusion compartment diameter was 188.5 ± 7.3 nm (n = 96; *, P ≤ 0.0001). The error bars show SEM. Bars: (A and B) 100 nm.
Mentions: Ultrastructural examination of the morphology of donor/acceptor membranes before a fusion reaction revealed the presence of consistently sized membrane-bound compartments (mean diameter, 58.3 ± 1.7 nm; Fig. 3Figure 3.

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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