Transmembrane organization of yeast syntaxin-analogue Sso1p.
Membrane fusion in secretory pathways is thought to be mediated by SNAREs. It is proposed that membrane fusion transits through hemifusion, a condition in which the outer leaflets of the bilayers are mixed, but the inner leaflets are not. Hemifusion then proceeds to the fusion pore that connects the two internal contents. It is believed that the transmembrane domains (TMDs) of the fusion proteins play an essential role in the transition from hemifusion to the fusion pore. In this work, the structure, dynamics, and membrane topology of the TMD of Sso1p, a target membrane (t-) SNARE involved in the trafficking from Golgi to plasma membrane in yeast, was investigated using site-directed spin labeling and EPR spectroscopy. The EPR analysis of spin-labeled mutants showed that the TMD of Sso1p is a well-defined membrane spanning alpha-helix. The results also indicate that there is an equilibrium between the monomers and the oligomers. The oligomerization is mainly mediated through the interaction at the N-terminal half of the TMD, whereas the C-terminal half is free of the tertiary interaction. Additionally, the isotropic hyperfine splitting values were examined for nitroxide-scanning mutants, and it was found that the hyperfine splitting values show a V-shaped profile across the bilayer. Thus, hyperfine splitting may be used as an additional parameter to measure bilayer immersion depths of nitroxide.
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Yinghui
Y
Zhang
Yeon-Kyun
YK
Shin
D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D008561:Membrane Fusion / Q000502:physiology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D013113:Spin Labels
D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C084006:SSO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013113:Spin Labels