PaperClip DNA Assembly: Reduce, Reuse, Recycle.

Creating DNA constructs is a basic and fundamental step in molecular and synthetic biology. While prices for gene synthesis are decreasing, it is still more economical in most cases to assemble constructs from a library of components (Parts). Many methods for DNA assembly are available, but most require either a fixed and inflexible format for the construct, with all Parts first being cloned in specific donor plasmids, or remaking Parts with new homology ends for each specific assembly reaction, requiring large numbers of single-use oligonucleotides. PaperClip assembly allows Parts stored in any format (linear PCR products or synthetic DNA, or cloned in any plasmid) to be used in totally flexible assembly reactions; up to 11 parts can be assembled in a single reaction, in any order, to give a linear or circular construct, and the oligonucleotides required in the assembly process can be reused in any subsequent assembly. In addition to constructing plasmids for bacterial transformation, PaperClip is also well suited to generate linear products for direct transfection of yeast, mammalian, or cyanobacterial cell lines. Thus, PaperClip offers a simple, flexible, and economical route to multipart assembly of constructs for a wide variety of purposes.

Voltage-sensor transitions of the inward-rectifying K+ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor.

The Kv-like (potassium voltage-dependent) K(+) channels at the plasma membrane, including the inward-rectifying KAT1 K(+) channel of Arabidopsis (Arabidopsis thaliana), are important targets for manipulating K(+) homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K(+) channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-directed mutagenesis to explore residues that are thought to form two electrostatic countercharge centers on either side of a conserved phenylalanine (Phe) residue within the S2 and S3 α-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the countercharge centers favored the open channel. Modeling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in the context of the effects on hydration of amino acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel.

Biodesalination: a case study for applications of photosynthetic bacteria in water treatment.

Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the next decades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in the oceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization of desalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for this purpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-salt reservoir within the saline water. The latter could be used as an ion exchanger through manipulation of transport proteins in the cell membrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for the exploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, genetic manipulation, ion transport, cell-water separation, process design, safety, and public acceptance.

Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic.

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual "handshaking" mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.

Ion transport, membrane traffic and cellular volume control.

Throughout their development, plants balance cell surface area and volume with ion transport and turgor. This balance lies at the core of cellular homeostatic networks and is central to the capacity to withstand abiotic as well as biotic stress. Remarkably, very little is known of its mechanics, notably how membrane traffic is coupled with osmotic solute transport and its control. Here we outline recent developments in the understanding of so-called SNARE proteins that form part of the machinery for membrane vesicle traffic in all eukaryotes. We focus on SNAREs active at the plasma membrane and the evidence for specialisation in enhanced, homeostatic and stress-related traffic. Recent studies have placed a canonical SNARE complex associated with the plasma membrane in pathogen defense, and the discovery of the first SNARE as a binding partner with ion channels has demonstrated a fundamental link to inorganic osmotic solute uptake. Work localising the channel binding site has now identified a new and previously uncharacterised motif, yielding important clues to a plausible mechanism coupling traffic and transport. We examine the evidence that this physical interaction serves to balance enhanced osmotic solute uptake with membrane expansion through mutual control of the two processes. We calculate that even during rapid cell expansion only a minute fraction of SNAREs present at the membrane need be engaged in vesicle traffic at any one time, a number surprisingly close to the known density of ion channels at the plant plasma membrane. Finally, we suggest a framework of alternative models coupling transport and traffic, and approachable through direct, experimental testing.

A molecular framework for coupling cellular volume and osmotic solute transport control.

Eukaryotic cells expand using vesicle traffic to increase membrane surface area. Expansion in walled eukaryotes is driven by turgor pressure which depends fundamentally on the uptake and accumulation of inorganic ions. Thus, ion uptake and vesicle traffic must be controlled coordinately for growth. How this coordination is achieved is still poorly understood, yet is so elemental to life that resolving the underlying mechanisms will have profound implications for our understanding of cell proliferation, development, and pathogenesis, and will find applications in addressing the mineral and water use by plants in the face of global environmental change. Recent discoveries of interactions between trafficking and ion transport proteins now open the door to an entirely new approach to understanding this coordination. Some of the advances to date in identifying key protein partners in the model plant Arabidopsis and in yeast at membranes vital for cell volume and turgor control are outlined here. Additionally, new evidence is provided of a wider participation among Arabidopsis Kv-like K(+) channels in selective interaction with the vesicle-trafficking protein SYP121. These advances suggest some common paradigms that will help guide further exploration of the underlying connection between ion transport and membrane traffic and should transform our understanding of cellular homeostasis in eukaryotes.

A novel motif essential for SNARE interaction with the K(+) channel KC1 and channel gating in Arabidopsis.

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 protein-protein 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.

A tripartite SNARE-K+ channel complex mediates in channel-dependent K+ nutrition in Arabidopsis.

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 inward-rectifying 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.

A protein interaction node at the neurotransmitter release site: domains of Aczonin/Piccolo, Bassoon, CAST, and rim converge on the N-terminal domain of Munc13-1.

Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.

AtIREG2 encodes a tonoplast transport protein involved in iron-dependent nickel detoxification in Arabidopsis thaliana roots.

Iron acquisition in Arabidopsis depends mainly on AtIRT1, a Fe2+ transporter in the plasma membrane of root cells. However, substrate specificity of AtIRT1 is low, leading to an excess accumulation of other transition metals in iron-deficient plants. In the present study we describe AtIREG2 as a nickel transporter at the vacuolar membrane that counterbalances the low substrate specificity of AtIRT1 and possibly other iron transport systems in iron-deficient root cells. AtIREG2 is co-regulated with AtIRT1 by the transcription factor FRU/FIT1, encodes a membrane protein, which has 10 putative transmembrane domains and shares homology with vertebrate Fe2+ exporters. Heterologous expression of AtIREG2 in various yeast mutants, however, did not demonstrate an iron transport function. Instead, expression in wild-type and nickel-sensitive cot1 yeast cells conferred enhanced tolerance to elevated concentrations of nickel at acidic pH. A role in vacuolar substrate transport was further supported by localization of AtIREG2-GFP fusion proteins to the tonoplast in Arabidopsis suspension cells and root cells of intact plants. Transgenic plants overexpressing AtIREG2 showed an increased tolerance to elevated concentrations of nickel, whereas T-DNA insertion lines lacking AtIREG2 expression were more sensitive to nickel, particularly under iron deficiency, and accumulated less nickel in roots. We therefore propose a role of AtIREG2 in vacuolar loading of nickel under iron deficiency and thus identify it as a novel component in the iron deficiency stress response.