False interaction of syntaxin 1A with a Ca2+-activated K+ channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein–protein interactions

Fletcher, S.M.; Bowden, Sarah E. H.; Marrion, Neil V.

doi:10.1016/s0028-3908(03)00049-2

Cited by 21 publications

(21 citation statements)

References 34 publications

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“…By contrast, immunoprecipitation with the Flag-tagged SYP111 did not yield a band detected by the anti-Myc antibody, although both extracts included similar levels of the epitope-tagged SNARE and KC1 proteins. Included in the protein gel blot analysis shown, the right-most lanes are of total protein precipitated from the final washes prior to elution, confirming the absence of nonspecific retention of the epitopes on the resin (Fletcher et al, 2003).…”

Section: Online)mentioning

confidence: 69%

“…This selectivity was evident in split-ubiquitin assays with yeast, in coimmunoprecipitation experiments after heterologous expression, and on expression as BiFC fusion constructs in Arabidopsis (Figures 1 and 2). Considered individually, each of these methods raises certain caveats with its application, whether in relation to non-native (heterologous) expression or to constitutive overexpression in vivo (Fletcher et al, 2003;Walter et al, 2004;Grefen et al, 2009). However, their common consensus in this singular result, and the lack of similar interactions among the closely related proteins tested in parallel, finds explanation only in the unique association between SYP121 and KC1.…”

Section: Syp121-kc1 Interaction Contributes To a Tripartite K + Channmentioning

confidence: 99%

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A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition inArabidopsis

et al. 2009

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A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K + channel subunit that assembles with different inward-rectifying K + channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K + channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K + channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inwardrectifying K + current in Arabidopsis root epidermal protoplasts as well as K + acquisition and growth in seedlings when channel-mediated K + uptake was limiting. That SYP121 should be important for gating of a K + channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE-ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K + uptake coordinately with membrane expansion for cell growth.

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Section: Online)mentioning

confidence: 69%

Section: Syp121-kc1 Interaction Contributes To a Tripartite K + Channmentioning

confidence: 99%

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition inArabidopsis

et al. 2009

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“…In both co-IP studies, it was ␣ 1C that was precipitated with RyR1. However, co-IP experiments must be interpreted with care because false positives are possible (Fletcher et al, 2003). Finally, RyRs have been observed to interact functionally with DHPRs in excised membrane patches of cerebellar granule cells (Chavis et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Dihydropyridine Receptors and Type 1 Ryanodine Receptors Constitute the Molecular Machinery for Voltage-Induced Ca²⁺Release in Nerve Terminals

et al. 2006

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“…The H3 a-helix also binds the CFTR Cl 2 channel (Naren et al, 1997;Ganeshan et al, 2003), although the association with vesicle fusion and possible roles for this interaction are less obvious. One difficulty in each of these examples rests with the amphipathic properties of the Q a -SNARE H3 a-helix: its seemingly promiscuous capacity for protein interaction has raised concerns about interpreting the physiological significance of SNARE binding with the channel proteins (Fletcher et al, 2003). It remains an unprecedented feature of SYP121, therefore, that its binding with KC1 depends not on the H3 or membrane-spanning a-helices, but on a previously unidentified motif isolated at the cytosolic N terminus of the SNARE and unique to SYP121.…”

Section: A Unique N-terminal Domain With Overlapping Functions?mentioning

confidence: 99%

A Novel Motif Essential for SNARE Interaction with the K+ Channel KC1 and Channel Gating inArabidopsis

et al. 2010

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The SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/ PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K + channel subunit KC1 and forms a tripartite complex with a second K + channel subunit, AKT1, to control channel gating and K + transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for proteinprotein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K + channel gating when heterologously expressed, and that rescue of KC1-associated K + current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE-ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.

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False interaction of syntaxin 1A with a Ca2+-activated K+ channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein–protein interactions

Cited by 21 publications

References 34 publications

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition inArabidopsis

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition inArabidopsis

Dihydropyridine Receptors and Type 1 Ryanodine Receptors Constitute the Molecular Machinery for Voltage-Induced Ca²⁺Release in Nerve Terminals

A Novel Motif Essential for SNARE Interaction with the K+ Channel KC1 and Channel Gating inArabidopsis

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False interaction of syntaxin 1A with a Ca2+-activated K+ channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein–protein interactions

Cited by 21 publications

References 34 publications

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition inArabidopsis

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition inArabidopsis

Dihydropyridine Receptors and Type 1 Ryanodine Receptors Constitute the Molecular Machinery for Voltage-Induced Ca2+Release in Nerve Terminals

A Novel Motif Essential for SNARE Interaction with the K+ Channel KC1 and Channel Gating inArabidopsis

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Dihydropyridine Receptors and Type 1 Ryanodine Receptors Constitute the Molecular Machinery for Voltage-Induced Ca²⁺Release in Nerve Terminals