High-affinity binding of the yeast cis-Golgi t-SNARE, Sed5p, to wild-type and mutant Sly1p, a modulator of transport vesicle docking.
Docking of ER-derived vesicles to the cis-Golgi compartment in yeast requires vesicle and target membrane receptors (v-SNAREs and t-SNAREs) and the GTPase Ypt1p. The t-SNARE Sed5p is complexed with Sly1p in vivo. The mutant form Sly1-20p rescues Ypt1p-lacking cells from lethality, suggesting an inhibitory function of Sly1p in v-SNARE/t-SNARE interaction. Using surface plasmon resonance spectroscopy, we found that Sed5p binds Sly1p and Sly1-20p with equally high affinity (K(D) = 5.13 x 10(-9) M and 4.74 x 10(-9) M, respectively). Deletion studies show that the N-terminal half of Sly1p rather than the C-terminus (harbouring the E532K substitution in Sly1-20p) is most critical for its binding to Sed5p. These data appear to argue for an active rather than an inhibitory role of Sly1p in vesicle docking.
Grabowski
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Max Planck Institute for Biophysical Chemistry, Department of Molecular Genetics, Göttingen, Germany.
Gallwitz
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D
D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D051938:Munc18 Proteins; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C072520:SLY1 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C077554:Sed5 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C098498:Ytp1 protein, S cerevisiae