VAMP721 Conformations Unmask an Extended Motif for K+ Channel Binding and Gating Control.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a major role in membrane fusion and contribute to cell expansion, signaling, and polar growth in plants. The SNARE SYP121 of Arabidopsis thaliana that facilitates vesicle fusion at the plasma membrane also binds with, and regulates, K+channels already present at the plasma membrane to affect K+uptake and K+-dependent growth. Here, we report that its cognate partner VAMP721, which assembles with SYP121 to drive membrane fusion, binds to the KAT1 K+channel via two sites on the protein, only one of which contributes to channel-gating control. Binding to the VAMP721 SNARE domain suppressed channel gating. By contrast, interaction with the amino-terminal longin domain conferred specificity on VAMP721 binding without influencing gating. Channel binding was defined by a linear motif within the longin domain. The SNARE domain is thought to wrap around this structure when not assembled with SYP121 in the SNARE complex. Fluorescence lifetime analysis showed that mutations within this motif, which suppressed channel binding and its effects on gating, also altered the conformational displacement between the VAMP721 SNARE and longin domains. The presence of these two channel-binding sites on VAMP721, one also required for SNARE complex assembly, implies a well-defined sequence of events coordinating K+uptake and the final stages of vesicle traffic. It suggests that binding begins with VAMP721, and subsequently with SYP121, thereby coordinating K+channel gating during SNARE assembly and vesicle fusion. Thus, our findings also are consistent with the idea that the K+channels are nucleation points for SNARE complex assembly.
Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
Ben
0000-0003-4473-9184
B
Zhang
Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
Rucha
0000-0001-6876-4099
R
Karnik
Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
Sakharam
0000-0003-1918-0673
S
Waghmare
Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
Naomi
0000-0002-1873-4286
N
Donald
Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ, United Kingdom michael.blatt@glasgow.ac.uk.
Michael R
0000-0003-1361-4645
MR
Blatt
D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015640:Ion Channel Gating / Q000502:physiology*; D008561:Membrane Fusion; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D017510:Protein Folding; D050765:Qa-SNARE Proteins / Q000378:metabolism; D050683:R-SNARE Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D029681:Arabidopsis Proteins; C501210:KAT1 protein, Arabidopsis; C479425:PEN1 protein, Arabidopsis; D024661:Potassium Channels, Inwardly Rectifying; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; C570049:VAMP721 protein, Arabidopsis; D014443:Tyrosine