Abstract
SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) assembly may promote intracellular membrane fusion, an essential process for vesicular transport in cells. Core complex formation between vesicle-associated SNARE and target membrane SNARE perhaps drives the merging of two membranes into a single bilayer. Using spin-labeling EPR, trans-SNARE complex formation was monitored "locally" at four different core locations of recombinant yeast SNAREs, which are individually reconstituted into phospholipid vesicles. The results indicate that the time scales of core formation are virtually the same at all four locations throughout the core region, indicating the possibility of a single step core assembly, which appears to be somewhat different from what has been postulated by the "zipper" model. The EPR data were then compared with the kinetics of the lipid mixing measured with the fluorescence assay. The analysis suggests that SNARE core assembly occurs on a much faster time scale than the lipid mixing, providing a new insight into the timing of individual events in SNARE-induced membrane fusion.
Highlights
Progress has been made in understanding the structure and the function of the SNARE complex
The results indicate that the time scales of core formation are virtually the same at all four locations throughout the core region, indicating the possibility of a single step core assembly, which appears to be somewhat different from what has been postulated by the “zipper” model
With site-directed spin labeling, conformational changes can be monitored “locally” at various locations of the polypeptide chain, providing a chance to resolve steps involved in the zippering process of SNARE complex formation
Summary
Plasmid Construction and Site-directed Mutagenesis—DNA sequences encoding Sso1pH3 (amino acids 185–265), Sso1pHT (amino acids 185–290), Snc2pS (amino acids 1–93), and Snc2pF (amino acids 1–115) were inserted into the pGEX-KG vector between EcoRI and HindIII sites as N-terminal glutathione S-transferase (GST) fusion proteins. Sec9c (amino acids 401– 651) was inserted into pET-24b(ϩ) between NdeI and XhoI sites as a C-terminal His6-tagged protein. A QuikChange site-directed mutagenesis kit (Stratagene) was used to generate all mutants, and DNA sequences were confirmed by the Iowa State University DNA Sequencing Facility. The cells were grown at 37 °C in LB medium with glucose (2 g/liter), ampicillin (100 g/ml), and chloramphenicol (25 g/ml) until the A600 reached 0.6 – 0.8. The cells were grown further for 4 more hours at 22 °C. The cell pellets were collected by centrifugation at 6000 rpm for 10 min
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