SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs - PubMed (original) (raw)

SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs

Thorsten Lang et al. J Cell Biol. 2002.

Abstract

During neuronal exocytosis, the vesicle-bound soluble NSF attachment protein (SNAP) receptor (SNARE) synaptobrevin 2 forms complexes with the plasma membrane-bound SNAREs syntaxin 1A and SNAP25 to initiate the fusion reaction. However, it is not known whether in the native membrane SNAREs are constitutively active or whether they are unable to enter SNARE complexes unless activated before membrane fusion. Here we used binding of labeled recombinant SNAREs to inside-out carrier supported plasma membrane sheets of PC12 cells to probe for the activity of endogenous SNAREs. Binding was specific, saturable, and depended on the presence of membrane-resident SNARE partners. Our data show that virtually all of the endogenous syntaxin 1 and SNAP-25 are highly reactive and readily form SNARE complexes with exogenously added SNAREs. Furthermore, complexes between endogenous SNAREs were not detectable when the membranes are freshly prepared, but they slowly form upon prolonged incubation in vitro. We conclude that the activity of membrane-resident SNAREs is not downregulated by control proteins but is constitutively active even if not engaged in fusion events.

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Figures

Figure 1.

Figure 1.

Syntaxin 1A binds to endogenous SNAP-25 on inside-out sheets of plasma membrane derived from PC12 cells. (A and B) Membrane sheets were reacted for 50 min at 37°C with syntaxin 1A(1–262)–Alexa594 (red channel) and then washed, fixed, and immunostained for SNAP-25 (green channel). (C and D) Magnified views from A and B. (E) Overlap from C and D. Circles indicate identical pixel locations. (F) On membrane sheets such as shown in A and B the staining intensity of random images in the green channel was measured and plotted against the staining intensity in the red channel, showing a linear correlation. (G) Effect of SNAP-25 cleavage by 2 μM of BoNT/E and/or Cl 71.1 (antibody specific for SNAP-25) on syntaxin binding, measured as above. Dilution of the antibody as indicated. (H) Effect of antibodies against rab3 (Cl 42.1), synaptobrevin 2 (Cl 69.1), and SNAP-25 (Cl 71.1) on syntaxin binding. (I) Binding of syntaxin in the presence of 2 μM BoNT/C1 (cleaving syntaxin) and 2 μM TeNT (cleaving synaptobrevin).

Figure 4.

Figure 4.

Endogenous syntaxin 1 is quantitatively cleaved by BoNT/C1 on freshly isolated inside-out membrane sheets but not after prolonged incubation suggesting formation of SNARE core complexes over time. (A–D) Membrane sheets treated for 45 min in the presence (C and D) or absence (A and B) of 5 μM BoNT/C1 were immunostained for syntaxin 1A/B (A and C) and SNAP-25 (B and D). (E) Quantification of syntaxin-immunostaining intensity on membrane sheets as shown in A and C and membrane sheets that were incubated for 1 h before BoNT/C1 treatment. Binding of syntaxin 1A–Alexa594 (F) and synaptobrevin 2–Alexa488 (G) to membrane sheets that were preincubated at 37°C in buffer for 0, 10, 30, or 60 min before addition of the labeled proteins.

Figure 2.

Figure 2.

Synaptobrevin 2 binds to endogenous syntaxin clusters on inside-out membrane sheets but also requires endogenous SNAP-25 for binding. (A and B) Membrane sheets were produced from cells expressing syntaxin 1A-GFP (green channel), and then reacted with synaptobrevin 2–Alexa594 (red channel). (C and D) Magnified views from A and B. (E) Overlap from C and D. (F) Plotting green against red fluorescence intensity from membrane sheets as shown in A and B. (G) Synaptobrevin 2 binding depends both on intact syntaxin 1 and SNAP-25, as it is inhibited by membrane treatment with either 2 μM BoNT/C1 or BoNT/E. (H) Membrane sheets were reacted with syntaxin 1A–Alexa594 for 60 min, washed two times at room temperature in BSA-KGlu buffer, and then reacted for 50 min with synaptobrevin 2–Alexa488. In this experiment, the measured green fluorescence intensity is an underestimate due to FRET occurring from the green to the red label. Note that in the experiments shown in G and H, nontransfected cells were used.

Figure 3.

Figure 3.

Syntaxin 1A–Alexa594 and synaptobrevin 2–Alexa488 form SNARE complexes on inside-out membrane sheets. (A–C) Membrane sheets reacted simultaneously with syntaxin 1A–Alexa594 (red channel) and synaptobrevin 2–Alexa488 (green channel). (D and E) NSF removes bound syntaxin 1 and synaptobrevin 2 from inside-out membranes. Membrane sheets reacted for 50 min with either synaptobrevin 2–Alexa488 (D) or syntaxin 1A–Alexa594 (E) were incubated with 0.5 μg/μl αSNAP and 0.1 μg/μl NSF during the last 10 min of the binding reaction, either in the presence of ATP or in the absence of ATP (including 1 mM EDTA). (F) Membrane sheets were reacted with syntaxin 1A–Alexa594 and synaptobrevin 2–Alexa488 either together (left) or alone (middle, right) and viewed in the green, red or the FRET channel. When both labeled proteins are bound a strong signal is detected in the FRET channel.

Figure 5.

Figure 5.

Binding of exogenous syntaxin 1A is not affected by syntaxin overexpression. Syntaxin 1A–Alexa594 binding was measured on membrane sheets from cells expressing syntaxin 1A–GFP. (A) Syntaxin 1A–GFP (green channel). (B) Syntaxin 1A–Alexa594 (red channel). (C and D) Magnified views from A and B. (E) Overlap from C and D. (F) Plotting green against red fluorescence intensity in random images obtained from membrane sheets as shown in A and B.

Figure 6.

Figure 6.

Working model: in the native plasma membrane the SNAREs are reactive. When isolated plasma membrane sheets age, the reactive endogenous SNAREs form binary and ternary complexes. Reactive endogenous SNAREs also form SNARE complexes with added recombinant SNAREs.

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