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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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EGF is transported through endocytic compartments after internalization. (A) Colocalization of early endosomes with EGF-TMR in HeLa cells. The distribution of EEA1 (A, green), EGF-TMR (B, red), and the merged images (C) shows colocalization of EEA1-labeled early endosomes and EGF-TMR. (B) Percentage of EEA1-labeled early endosomes colocalized with EGF-TMR–containing vesicles after various incubation times. (C) Percentage of rab 7–labeled late endosomes colocalized with EGF-TMR–containing vesicles at various chase times after a 15-min pulse of EGF-TMR. (D) Percentage of LAMP 1/2–labeled lysosomes colocalized with EGF-TMR–containing vesicles at various chase times after a 15-min pulse of EGF-TMR. The error bars in B–D show SEM. Bar, 8 μm.
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fig1: EGF is transported through endocytic compartments after internalization. (A) Colocalization of early endosomes with EGF-TMR in HeLa cells. The distribution of EEA1 (A, green), EGF-TMR (B, red), and the merged images (C) shows colocalization of EEA1-labeled early endosomes and EGF-TMR. (B) Percentage of EEA1-labeled early endosomes colocalized with EGF-TMR–containing vesicles after various incubation times. (C) Percentage of rab 7–labeled late endosomes colocalized with EGF-TMR–containing vesicles at various chase times after a 15-min pulse of EGF-TMR. (D) Percentage of LAMP 1/2–labeled lysosomes colocalized with EGF-TMR–containing vesicles at various chase times after a 15-min pulse of EGF-TMR. The error bars in B–D show SEM. Bar, 8 μm.

Mentions: After increasing periods of chase time EGF-TMR–labeled cells were immunolabeled with markers for the early endosome (EEA-1), late endosome (rab 7), and lysosome (LAMP1/2). By quantifying the amount of overlap between the two signals we generated a time course of EGF-TMR movement through the endocytic pathway that we used as the optimal labeling time for the various compartments. When HeLa cells are incubated with EGF-TMR for 15 min, the predominant localization of the EGF-TMR labeling was in an EEA-1–positive structure, putatively an early endosome (Fig. 1, A and B)Figure 1.


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)

EGF is transported through endocytic compartments after internalization. (A) Colocalization of early endosomes with EGF-TMR in HeLa cells. The distribution of EEA1 (A, green), EGF-TMR (B, red), and the merged images (C) shows colocalization of EEA1-labeled early endosomes and EGF-TMR. (B) Percentage of EEA1-labeled early endosomes colocalized with EGF-TMR–containing vesicles after various incubation times. (C) Percentage of rab 7–labeled late endosomes colocalized with EGF-TMR–containing vesicles at various chase times after a 15-min pulse of EGF-TMR. (D) Percentage of LAMP 1/2–labeled lysosomes colocalized with EGF-TMR–containing vesicles at various chase times after a 15-min pulse of EGF-TMR. The error bars in B–D show SEM. Bar, 8 μm.
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Related In: Results  -  Collection

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fig1: EGF is transported through endocytic compartments after internalization. (A) Colocalization of early endosomes with EGF-TMR in HeLa cells. The distribution of EEA1 (A, green), EGF-TMR (B, red), and the merged images (C) shows colocalization of EEA1-labeled early endosomes and EGF-TMR. (B) Percentage of EEA1-labeled early endosomes colocalized with EGF-TMR–containing vesicles after various incubation times. (C) Percentage of rab 7–labeled late endosomes colocalized with EGF-TMR–containing vesicles at various chase times after a 15-min pulse of EGF-TMR. (D) Percentage of LAMP 1/2–labeled lysosomes colocalized with EGF-TMR–containing vesicles at various chase times after a 15-min pulse of EGF-TMR. The error bars in B–D show SEM. Bar, 8 μm.
Mentions: After increasing periods of chase time EGF-TMR–labeled cells were immunolabeled with markers for the early endosome (EEA-1), late endosome (rab 7), and lysosome (LAMP1/2). By quantifying the amount of overlap between the two signals we generated a time course of EGF-TMR movement through the endocytic pathway that we used as the optimal labeling time for the various compartments. When HeLa cells are incubated with EGF-TMR for 15 min, the predominant localization of the EGF-TMR labeling was in an EEA-1–positive structure, putatively an early endosome (Fig. 1, A and B)Figure 1.

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