Toru Ide

Uncovering the Protein Translocon at the Chloroplast Inner Envelope Membrane

Chloroplast Translocon Revealed Protein translocation across biological membranes requires supramolecular complexes, called translocons. Chloroplasts require translocons in their double-envelope membranes to import thousands of nucleus-encoded proteins synthesized in the cytosol. However, the identity of the translocon at the inner envelope of the chloroplast (TIC) has long been a matter of debate; two proteins, Tic20 and Tic110, have been proposed to be central to protein translocation across the inner envelope membrane. Using transgenic Arabidopsis plants expressing a tagged form of Tic20, Kikuchi et al. (p. 571) report the isolation of a 1-megadalton complex composed of Tic56, Tic100, and Tic214 involved in protein translocation across the inner envelope. Thorough in vitro biochemical and in vivo genetic experimentation suggest that the isolated translocon contains both nuclear- and organellar-encoded components. Tic110 was not part of the isolated translocon. The protein transport channel in the chloroplast inner envelope requires both nuclear- and organelle-encoded subunits. Chloroplasts require protein translocons at the outer and inner envelope membranes, termed TOC and TIC, respectively, to import thousands of cytoplasmically synthesized preproteins. However, the molecular identity of the TIC translocon remains controversial. Tic20 forms a 1-megadalton complex at the inner membrane and directly interacts with translocating preproteins. We purified the 1-megadalton complex from Arabidopsis, comprising Tic20 and three other essential components, one of which is encoded by the enigmatic open reading frame ycf1 in the chloroplast genome. All four components, together with well-known TOC components, were found stoichiometrically associated with different translocating preproteins. When reconstituted into planar lipid bilayers, the purified complex formed a preprotein-sensitive channel. Thus, this complex constitutes a general TIC translocon.

Automated Parallel Recordings of Topologically Identified Single Ion Channels

Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non‐cargo proteins during COPII vesicle formation using single‐molecule microscopy combined with an artificial planar lipid bilayer. Single‐molecule analysis showed that the Sar1p–Sec23/24p‐cargo complex, but not the Sar1p–Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non‐cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non‐cargo proteins from the COPII vesicles.

Development of an Experimental Apparatus for Simultaneous Observation of Optical and Electrical Signals from Single Ion Channels

We have developed an experimental apparatus for the simultaneous measurement of optical and electrical properties of single ion channels. The microscope designed for single-molecule detection was combined with the artificial bilayer single-channel recording system. The artificial membranes were formed horizontally in an aqueous environment or on a thin agarose layer. Single molecules in the bilayer were observed under an epi-fluorescence or an objective-type total internal reflection fluorescence micoscope. Minute currents across the bilayers were measured by a patch clamp amplifier under voltage clamped conditions. The apparatus developed in this study was sensitive enough to detect the optical signals from single-fluorophores in the bilayer simultaneously with the single-channel current recording. Using this apparatus, the following results were obtained: (i) Brownian motion of single-lipid molecules in the bilayer was observed. (ii) Cy3-alamethicin molecules were incorporated into the bilayer and the fluorescence image was recorded simultaneously with the channel current recording at the single-channel level. (iii) The liposome fusion process to the bilayer could be observed at the single molecule level. (iv) Various types of channels could be incorporated into the bilayers on agarose. The properties of the channels were identical to those determined in the bilayer formed in an aqueous environment.

Lipid bilayers at the gel interface for single ion channel recordings

Single-channel recording using artificial lipid bilayers is along with the patch-clamp technique a very powerful tool to physiologically and pharmacologically study ion channels. It is particularly advantageous in studying channels that are technically difficult to access with a patch pipet. However, the fragility of the bilayers and the difficulty to incorporate ion channels into them significantly compromises measurement efficiency. We have developed a novel method for forming artificial lipid bilayers on a hydrogel surface that significantly improves the measurement efficiency. Bilayers formed almost instantly (<1 s) and were able to incorporate various types of ion channel proteins within a short time (<30 s) enabling multichannel measurements. These results indicate that this method can potentially be applied to developing high-throughput screening devices for drug design.

Rearrangements in the KcsA Cytoplasmic Domain Underlie Its Gating

A change of cytosolic pH 7 to 4 opens the bacterial potassium channel KcsA. However, the overall gating mechanism leading to channel opening, especially the contribution of the cytoplasmic domain, remains unsolved. Here we report that deletion of the cytoplasmic domain resulted in changes in channel conductance and gating behavior at pH 4 without channel opening at pH 7. To probe for rearrangements in the cytoplasmic domain during channel opening, amino acid residues were substituted with cysteines and labeled with a fluorophore (tetramethylrhodamine maleimide) that exhibits increased fluorescence intensity upon transfer from a hydrophilic to hydrophobic environment. In all cases channel open probability (Po) was ∼1 at pH 4 and ∼0 at pH 7. Major increases in fluorescence intensity were observed for tetramethylrhodamine maleimide-labeled residues in the cytoplasmic domain as pH changed from 7 to 4, which suggests the fluorophores shifted from a hydrophilic to hydrophobic environment. Dipicrylamide, a lipid soluble quencher, reduced the fluorescence intensities of labeled residues in the cytosolic domain at pH 4. These results reveal that a decrease in pH introduces major conformational rearrangements associated with channel opening in the KcsA cytoplasmic domain.

A glass fiber sheet-based electroosmotic lateral flow immunoassay for point-of-care testing.

We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.

Role of the KcsA Channel Cytoplasmic Domain in pH-Dependent Gating

The KcsA channel is a representative potassium channel that is activated by changes in pH. Previous studies suggested that the region that senses pH is entirely within its transmembrane segments. However, we recently revealed that the cytoplasmic domain also has an important role, because its conformation was observed to change dramatically in response to pH changes. Here, to investigate the effects of the cytoplasmic domain on pH-dependent gating, we made a chimera mutant channel consisting of the cytoplasmic domain of the KcsA channel and the transmembrane region of the MthK channel. The chimera showed a pH dependency similar to that of KcsA, indicating that the cytoplasmic domain can act as a pH sensor. To identify how this region detects pH, we substituted certain cytoplasmic domain amino acids that are normally negatively charged at pH 7 for neutral ones in the KcsA channels. These mutants opened independently of pH, suggesting that electrostatic charges have a major role in the cytoplasmic domains ability to sense and respond to pH.

Mast cell degranulating peptide forms voltage gated and cation-selective channels in lipid bilayers

Mast cell degranulating peptide, a toxin of bee venom, is a polypeptide composed of 22 amino acids. Exposure of the asolectin bilayer to the peptide results in the formation of channels that are more permeable to K+ than Cl-. These channels are activated when the voltage of the cis side, to which the peptide is added, is made positive.

A voltage- and K+-dependent K+ channel from a membrane fraction enriched in contractile vacuole of Dictyostelium discoideum.

We obtained a membrane fraction enriched in the contractile vacuole by aqueous-polymer two-phase partitioning and its channel activities were analysed by incorporating it into artificial planar lipid bilayers. In asymmetrical KCl solutions (cis, 300 mM/100 mM, trans), we observed single-channel currents of a highly K(+)-selective channel with slope conductance of 102 pS and reversal potential of -20.4 mV, which corresponded to PK+/PCl- = 7. They showed bursts separated by infrequent quiescent periods. At 0 mV the mean open time was 2.0 ms. Among monovalent cations, Na+ and Li+ were impermeable, whereas Rb+ showed permeability equivalent to that of K+, although the unitary conductance was apparently reduced when the current flowed from the Rb+ containing side, suggesting that Rb+ is a permeant blocking ion. The open probability within bursts remained constant at approx.0.6 as long as the holding potential was positive on the cis side with respect to the trans side, but it decreased to 0 at negative potential. This channel was blocked by submillimolar concentrations of quinine and 30 mM TEA+. The open probability-voltage relationship showed a striking dependency on the KCl concentration on either side. This channel may play a role in water transport in this organelle.