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

In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR)-encoded immunity in Type I systems relies on the Cascade (CRISPR-associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4-Cas (CRISPR-associated) system (St-CRISPR4-Cas), we isolated an effector complex (St-Cascade) containing 61-nucleotide CRISPR RNA (crRNA). We show that St-Cascade, guided by crRNA, binds in vitro to a matching proto-spacer if a proto-spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (-1 position) of the proto-spacer. In the presence of a correct PAM, St-Cascade binding to the target DNA generates an R-loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R-loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St-CRISPR4-Cas and other Type I systems

Gating control and K+ uptake by the KAT1 K+ channel leaveraged through membrane anchoring of the trafficking protein SYP121

Vesicle traffic is tightly coordinated with ion transport for plant cell expansion through physical interactions between subsets of vesicle‐trafficking (so‐called SNARE) proteins and plasma membrane Kv channels, including the archetypal inward‐rectifying K+ channel, KAT1 of Arabidopsis. Ion channels open and close rapidly over milliseconds, whereas vesicle fusion events require many seconds. Binding has been mapped to conserved motifs of both the Kv channels and the SNAREs, but knowledge of the temporal kinetics of their interactions, especially as it might relate to channel gating and its coordination with vesicle fusion remains unclear. Here we report that the SNARE SYP121 promotes KAT1 gating through a persistent interaction that alters the stability of the channel, both in its open and closed states. We show, too, that SYP121 action on the channel open state requires SNARE anchoring in the plasma membrane. Our findings indicate that SNARE binding confers a conformational bias that encompasses the microscopic kinetics of channel gating, with leverage applied through the SNARE anchor in favor of the open channel

RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions

Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated (cas) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a "seed sequence" at the 5' end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems

K+ channel and SEC11 binding exchange regulates SNARE assembly for secretory traffic

Cell expansion requires that ion transport and secretory membrane traffic operate in concert. Evidence from Arabidopsis (Arabidopsis thaliana) indicates that such coordination is mediated by physical interactions between subsets of so-called SNARE proteins, which drive the final stages of vesicle fusion, and K+ channels that facilitate uptake of the cation to maintain cell turgor pressure as the cell expands. However, the sequence of SNARE binding with the K+ channels and its interweaving within the events of SNARE complex assembly for exocytosis has remained unclear. We have combined protein-protein interaction and electrophysiological analyses to resolve the binding interactions of the hetero-oligomeric associations. We find that the RYxxWE motif, located within the voltage sensor of the K+ channels, is a nexus for multiple SNARE interactions. Of these, K+ channel binding and its displacement of the regulatory protein SEC11 is critical to prime the Qa-SNARE SYP121. Our results indicate a stabilizing role for the Qbc-SNARE SNAP33 in Qa-SNARE transition to SNARE complex assembly with the R-SNARE VAMP721. They also suggest that, on its own, the R-SNARE enters an anomalous binding mode with the channels, possibly as a fail-safe to ensure a correct binding sequence. Thus, we suggest that SYP121 binding to the K+ channels serves the role of a primary trigger to initiate assembly of the secretory machinery for exocytosis

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

SYNTAXIN OF PLANTS 132 underpins secretion of cargoes associated with salicylic acid signaling and pathogen defense

Secretory trafficking in plant cells is facilitated by SNARE (soluble N-ethylamide-sensitive factor attachment protein receptor) proteins that drive membrane fusion of cargo-containing vesicles. In Arabidopsis, SYNTAXIN OF PLANTS 132 (SYP132) is an evolutionarily ancient SNARE that functions with syntaxins SYP121 and SYP122 at the plasma membrane. Whereas SYP121 and SYP122 mediate overlapping secretory pathways, albeit with differences in their importance in plant-environment interactions, the SNARE SYP132 is absolutely essential for plant development and survival. SYP132 promotes endocytic traffic of the plasma membrane H+-ATPase AHA1 and aquaporin PIP2;1, and it coordinates plant growth and bacterial pathogen immunity through PATHOGENESIS-RELATED1 (PR1) secretion. Yet, little else is known about SYP132 cargoes. Here, we used advanced quantitative Tandem Mass Tagging (TMT) mass spectrometry (MS) combined with immunoblot assays to track native secreted cargo proteins in the leaf apoplast. We found that SYP132 supports a basal level of secretion in Arabidopsis leaves, and its overexpression influences salicylic acid (SA) and jasmonic acid (JA) defence-related cargoes including PR1, PR2, and PR5 proteins. Impairing SYP132 function also suppressed defence-related secretory traffic when challenged with the bacterial pathogen Pseudomonas syringae. Thus, we conclude that, in addition to its role in hormone-related H+-ATPase cycling, SYP132 influences basal plant immunity

Characterization and Quantification of RNA Post-transcriptional Modifications Using Stable Isotope Labeling of RNA in Conjunction with Mass Spectrometry Analysis

Mass spectrometry has emerged as an increasingly powerful tool for the identification and characterization of nucleic acids, in particular RNA post-transcriptional modifications. High mass accuracy instrumentation is often required to discriminate between compositional isomers of oligonucleotides. We have used stable isotope labeling (15N) of E. coli RNA in conjunction with mass spectrometry analysis of the combined heavy- and light-labeled RNA for the identification and quantification of oligoribonucleotides and post-transcriptional modifications. The number of nitrogen atoms in the oligoribonucleotide and fragment ions can readily be determined using this approach, enabling the discrimination between potential compositional isomers without the requirement of high mass accuracy mass spectrometers. In addition, the identification of specific fragment ions in both the unlabeled and labeled oligoribonucleotides can be used to gain further confidence in the assignment of RNA post-transcriptional modifications. Using this approach we have identified a range of post-transcriptional modifications of E. coli 16S rRNA. Furthermore, this method facilitates the rapid and accurate quantification of oligoribonucleotides, including cyclic phosphate intermediates and missed cleavages often generated from RNase digestions