Functional Analysis Of The SNARE Domain Of Q-sNARE Proteins Using Yeast Sporulation

In eukaryotic cells, vesicle fusion machinery including synaptic vesicle fusion is wellconserved, and Soluble NSF Attachment Protein Receptor (SNARE) proteins act as a centralpart of the machinery. For synaptic vesicle fusion, syntaxin-1and SNAP-25act as Q-SNAREs,and synaptobrevin acts as a R-SNARE. When neural signal reaches the pre-synaptic terminal,synaptic vesicles fuse with the plasma membrane through SNARE complex composed withsyntaxin-1, SNAP-25and synaptobrevin and release neurotransmitters, then the signal istransmitted to the post-synaptic cell.The regulation of this process is very strict and complex,which has not yet fully clear. And neuronal cells are not easy to handle, thus, not suitable forgenetic and drug screenings.SNAREs involved in PrM formation are homologues of synaptic SNAREs. On theprecursor vesicles of PrM, Sso1and Spo20are on one side of the apposing vesicle asQ-SNAREs, and either Snc1or Snc2work as a R-SNARE on the other side of the vesicle.Sso1, Spo20and Snc1or Snc2are yeast homologues of human syntaxin-1, SNAP-25andsynaptobrevin, respectively. Yeast sporulation is an ideal model system to manipulate andinvestigate vesicle fusion machinery. In this study, we reconstituted part of synapticQ-SNAREsyntaxin-1A and SNAP-25in S. cerevisiae. The main results are as follows:1. Sso1/syntaxin-1A chimera in which entire Sso1SNARE domain is replaced with thatof syntaxin-1A cannot complement sporulation deficiency of sso1mutant. To determinewhich residues are critical for Sso1function, we employed further chimera and mutationalanalyses and found that the Sso1/syntaxin-1A chimera gain the functionality by replacingAla220residue to Glu in the syntaxin-1A SNARE domain. We successfully constructed usefulrecombinant protein by changing A220to E.2. Mapping of the sporulation-specific functions in Spo20using fusions with SNAP-25indicates that sporulation needs both part of the two SNARE domains (before the centralamino acids Q). Modified Spo20into humanized chimera-change amino acids after Q of thefirst SNARE domain and355-397amino acid of the second SNARE domain to SNAP-25andobtained useful recombinant protein which can play a role in vesicle fusion process.3. Further study of the Sso1E218showed that this site has a high tolerance to themutation and the electronegativity is not essential for vesicle fusion. Overexpression of Sec1has no effect on sporulation efficiency of recombinant strains.The growth experiments of vegetative cells for sso1Δsso2Δstrain was consistent with theanalysis of sporulation, which can be used to verify the role of other recombinant proteins andinteraction between Sso1/syntaxin-1A (SNARE) and other proteins in vesicle fusion process.sso1Δsso2Δcan also be uesd to screen protein or pharmaceutical which interact withsyntaxin-1A.We believe this result will provide a good foundation for the subsequent establishof humanized SNARE system and related theoretical study.