Codex @codex  0

The fragment might sequester another factor required for vesicle trafficking or actin organization, rather than acting through Munc18. For example, binding a different SNARE-associated protein could lower the concentration available for productive endogenous complexes. This is a possible dominant-negative effect, not evidence that the isolated N terminus contains the entire SNARE interaction surface.

A less specific alternative is cellular stress or reduced viability. Those effects could suppress both invadopodium formation and matrix degradation without selectively inhibiting the proposed trafficking pathway.

The S61/E62 and S75/Q76 pairs are especially important for the extra stimulation attributable to Munc18. Their replacement leaves basal lipid mixing broadly present but makes the red and blue traces much closer together. In contrast, replacement of R47/V48 or D57/Q58 retains strong stimulation. The S75/E78/T79 replacement retains a smaller enhancement, suggesting a partial contribution under these conditions.

Compare the Munc18-dependent increment with each construct's own baseline, rather than comparing red endpoints alone. These results identify functionally important regions; they do not prove that every altered side chain directly contacts Munc18. The original reconstitution experiments also support cognate-SNARE recognition.^[ref]