Limits...
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

Related in: MedlinePlus

Characterization of a FRET-based assay measuring homotypic compartment fusion. (A) Fusion reactions were performed without the indicated component or on ice (0°C) to assess requirements for the reaction. There are significant differences between the complete homotypic fusion reactions and all other conditions in which a component has been omitted or altered (*, P ≤ 0.001) by ANOVA and Fisher test. Raw data after background subtraction are as follows. Early endosome fusion: no donor, 14.1 ± 3.5; no acceptor, 15.4 ± 5.0; no ATP, 37.3 ± 16.0; no cytosol, 28.3 ± 3.6; ice, 10.7 ± 4.3; and complete homotypic reaction, 302.5 ± 7.7. Late endosome fusion: no donor, 16.3 ± 3.4; no acceptor, 13.1 ± 12.8; no ATP, 15.6 ± 10.8; no cytosol, 26.6 ± 7.8; ice, 12.8 ± 4.8; and complete homotypic reaction, 312.1 ± 6.3. Lysosome fusion: no donor, 28.4 ± 1.4; no acceptor, 21.0 ± 6.2; no ATP, 23.9 ± 8.2; no cytosol, 24.3 ± 7.0; ice, 13.3 ± 1.2; and complete homotypic reaction, 325.3 ± 16.0. (B) Energy transfer is dependent on EGF-labeled donor and acceptor membranes. When both fluorescent tags are present on EGF and endosomes are labeled and fused a FRET signal occurs. However, labeling the acceptor population of endosomes with transferrin-TMR reduces the FRET signal to background levels (*, P ≤ 0.01). The error bars show SEM. (C) The optimal incubation time for fusion reactions was determined by examining the extent of fusion after incubating the reactions for various amounts of time at 37°C. The amount of fusion increased until 60 min of incubation, after which no further significant increase in fusion is observed. (D) The fusion reaction volume was increased to dilute the concentration of donor/acceptor membranes while the concentrations of ATP and cytosol were maintained at a constant level. A decrease in FRET signal was observed concomitant with an incremental increase in the reaction volume. (E) Syntaxin 13 and 7 affect homotypic endosome fusion. Syntaxin 13 inhibited early endosome homotypic fusion. Syntaxin 7 inhibited homotypic lysosome fusion. These data are consistent with previously published reports (see Introduction and Results). *, P ≤ 0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172712&req=5

fig2: Characterization of a FRET-based assay measuring homotypic compartment fusion. (A) Fusion reactions were performed without the indicated component or on ice (0°C) to assess requirements for the reaction. There are significant differences between the complete homotypic fusion reactions and all other conditions in which a component has been omitted or altered (*, P ≤ 0.001) by ANOVA and Fisher test. Raw data after background subtraction are as follows. Early endosome fusion: no donor, 14.1 ± 3.5; no acceptor, 15.4 ± 5.0; no ATP, 37.3 ± 16.0; no cytosol, 28.3 ± 3.6; ice, 10.7 ± 4.3; and complete homotypic reaction, 302.5 ± 7.7. Late endosome fusion: no donor, 16.3 ± 3.4; no acceptor, 13.1 ± 12.8; no ATP, 15.6 ± 10.8; no cytosol, 26.6 ± 7.8; ice, 12.8 ± 4.8; and complete homotypic reaction, 312.1 ± 6.3. Lysosome fusion: no donor, 28.4 ± 1.4; no acceptor, 21.0 ± 6.2; no ATP, 23.9 ± 8.2; no cytosol, 24.3 ± 7.0; ice, 13.3 ± 1.2; and complete homotypic reaction, 325.3 ± 16.0. (B) Energy transfer is dependent on EGF-labeled donor and acceptor membranes. When both fluorescent tags are present on EGF and endosomes are labeled and fused a FRET signal occurs. However, labeling the acceptor population of endosomes with transferrin-TMR reduces the FRET signal to background levels (*, P ≤ 0.01). The error bars show SEM. (C) The optimal incubation time for fusion reactions was determined by examining the extent of fusion after incubating the reactions for various amounts of time at 37°C. The amount of fusion increased until 60 min of incubation, after which no further significant increase in fusion is observed. (D) The fusion reaction volume was increased to dilute the concentration of donor/acceptor membranes while the concentrations of ATP and cytosol were maintained at a constant level. A decrease in FRET signal was observed concomitant with an incremental increase in the reaction volume. (E) Syntaxin 13 and 7 affect homotypic endosome fusion. Syntaxin 13 inhibited early endosome homotypic fusion. Syntaxin 7 inhibited homotypic lysosome fusion. These data are consistent with previously published reports (see Introduction and Results). *, P ≤ 0.01.

Mentions: Based on the optimal labeling conditions for each compartment (Fig. 1), fluorescently labeled early and late endosomal populations, as well as lysosomes, were isolated and used in fusion reactions. In the absence of donor or acceptor membranes, ATP, or cytosol, compartment fusion did not occur as visualized because of a lack of a TMR emission signal (580 nm) that is significantly above baseline after excitation of the Alexa fluorophore at 495 nm (Fig. 2Figure 2.


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)

Characterization of a FRET-based assay measuring homotypic compartment fusion. (A) Fusion reactions were performed without the indicated component or on ice (0°C) to assess requirements for the reaction. There are significant differences between the complete homotypic fusion reactions and all other conditions in which a component has been omitted or altered (*, P ≤ 0.001) by ANOVA and Fisher test. Raw data after background subtraction are as follows. Early endosome fusion: no donor, 14.1 ± 3.5; no acceptor, 15.4 ± 5.0; no ATP, 37.3 ± 16.0; no cytosol, 28.3 ± 3.6; ice, 10.7 ± 4.3; and complete homotypic reaction, 302.5 ± 7.7. Late endosome fusion: no donor, 16.3 ± 3.4; no acceptor, 13.1 ± 12.8; no ATP, 15.6 ± 10.8; no cytosol, 26.6 ± 7.8; ice, 12.8 ± 4.8; and complete homotypic reaction, 312.1 ± 6.3. Lysosome fusion: no donor, 28.4 ± 1.4; no acceptor, 21.0 ± 6.2; no ATP, 23.9 ± 8.2; no cytosol, 24.3 ± 7.0; ice, 13.3 ± 1.2; and complete homotypic reaction, 325.3 ± 16.0. (B) Energy transfer is dependent on EGF-labeled donor and acceptor membranes. When both fluorescent tags are present on EGF and endosomes are labeled and fused a FRET signal occurs. However, labeling the acceptor population of endosomes with transferrin-TMR reduces the FRET signal to background levels (*, P ≤ 0.01). The error bars show SEM. (C) The optimal incubation time for fusion reactions was determined by examining the extent of fusion after incubating the reactions for various amounts of time at 37°C. The amount of fusion increased until 60 min of incubation, after which no further significant increase in fusion is observed. (D) The fusion reaction volume was increased to dilute the concentration of donor/acceptor membranes while the concentrations of ATP and cytosol were maintained at a constant level. A decrease in FRET signal was observed concomitant with an incremental increase in the reaction volume. (E) Syntaxin 13 and 7 affect homotypic endosome fusion. Syntaxin 13 inhibited early endosome homotypic fusion. Syntaxin 7 inhibited homotypic lysosome fusion. These data are consistent with previously published reports (see Introduction and Results). *, P ≤ 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Characterization of a FRET-based assay measuring homotypic compartment fusion. (A) Fusion reactions were performed without the indicated component or on ice (0°C) to assess requirements for the reaction. There are significant differences between the complete homotypic fusion reactions and all other conditions in which a component has been omitted or altered (*, P ≤ 0.001) by ANOVA and Fisher test. Raw data after background subtraction are as follows. Early endosome fusion: no donor, 14.1 ± 3.5; no acceptor, 15.4 ± 5.0; no ATP, 37.3 ± 16.0; no cytosol, 28.3 ± 3.6; ice, 10.7 ± 4.3; and complete homotypic reaction, 302.5 ± 7.7. Late endosome fusion: no donor, 16.3 ± 3.4; no acceptor, 13.1 ± 12.8; no ATP, 15.6 ± 10.8; no cytosol, 26.6 ± 7.8; ice, 12.8 ± 4.8; and complete homotypic reaction, 312.1 ± 6.3. Lysosome fusion: no donor, 28.4 ± 1.4; no acceptor, 21.0 ± 6.2; no ATP, 23.9 ± 8.2; no cytosol, 24.3 ± 7.0; ice, 13.3 ± 1.2; and complete homotypic reaction, 325.3 ± 16.0. (B) Energy transfer is dependent on EGF-labeled donor and acceptor membranes. When both fluorescent tags are present on EGF and endosomes are labeled and fused a FRET signal occurs. However, labeling the acceptor population of endosomes with transferrin-TMR reduces the FRET signal to background levels (*, P ≤ 0.01). The error bars show SEM. (C) The optimal incubation time for fusion reactions was determined by examining the extent of fusion after incubating the reactions for various amounts of time at 37°C. The amount of fusion increased until 60 min of incubation, after which no further significant increase in fusion is observed. (D) The fusion reaction volume was increased to dilute the concentration of donor/acceptor membranes while the concentrations of ATP and cytosol were maintained at a constant level. A decrease in FRET signal was observed concomitant with an incremental increase in the reaction volume. (E) Syntaxin 13 and 7 affect homotypic endosome fusion. Syntaxin 13 inhibited early endosome homotypic fusion. Syntaxin 7 inhibited homotypic lysosome fusion. These data are consistent with previously published reports (see Introduction and Results). *, P ≤ 0.01.
Mentions: Based on the optimal labeling conditions for each compartment (Fig. 1), fluorescently labeled early and late endosomal populations, as well as lysosomes, were isolated and used in fusion reactions. In the absence of donor or acceptor membranes, ATP, or cytosol, compartment fusion did not occur as visualized because of a lack of a TMR emission signal (580 nm) that is significantly above baseline after excitation of the Alexa fluorophore at 495 nm (Fig. 2Figure 2.

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