The fusion pore connects the intravesicular lumen to the extracellular space, allowing for release of vesicular contents ( Breckenridge and Almers, 1987).
Many types of cells release specific molecules stored in intracellular vesicles by exocytosis via the formation of a fusion pore. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus.
#Positively charged amino acids Patch
To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25Δ9) showed changed fusion pore properties ( Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. Furthermore, we explored the presence of this cluster at the N terminus of the mitochondrial proteome and propose a set of precursors whose proper localization depends on both αβ′-NAC and Sam37.SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules. Our results reveal the presence of a positively charged amino acid cluster in the MTS of select mitochondrial precursors, such as Oxa1 and Fum1, which are crucial for their recognition by αβ′-NAC. We used targeting signals of different mitochondrial proteins (αβ′-NAC-dependent Oxa1 and αβ′-NAC-independent Mdm38) and fused them to GFP to study their intracellular localization by biochemical and microscopy methods, and in addition followed their import kinetics in vivo. In this work, we aimed to detect the region in the MTS of mitochondrial precursors relevant for their recognition by αβ′-NAC during their sorting to the mitochondria. We have previously described that the mitochondrial outer membrane protein Sam37 interacts with αβ′-NAC and together promote the import of specific mitochondrial precursor proteins. The cytosolic chaperone nascent polypeptide–associated complex (NAC), which in yeast is represented as the two different heterodimers αβ-NAC and αβ′-NAC, has been proposed to be involved during the early steps of mitochondrial protein targeting. However, the early steps of mitochondrial protein targeting remain undeciphered. For a subset of mitochondrial proteins, a signal sequence at the N terminus (matrix-targeting sequence ) is recognized by protein complexes to ensure their proper translocation into the organelle.
A major challenge in eukaryotic cells is the proper distribution of nuclear-encoded proteins to the correct organelles.
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