Tailoring baker's yeast Saccharomyces cerevisiae for functional testing of channelrhodopsin.

Channelrhodopsin 2 (ChR2) and its variants are the most frequent tools for remote manipulation of electrical properties in cells via light. Ongoing attempts try to enlarge their functional spectrum with respect to ion selectivity, light sensitivity and protein trafficking by mutations, protein engineering and environmental mining of ChR2 variants. A shortcoming in the required functional testing of large numbers of ChR2 variants is the lack of an easy screening system.

Novel auto-selection systems for transformation selection of Saccharomyces cerevisiae in rich complex media.

The most widely used strategy for selection of yeast transformed with episomal plasmids comprises the use of auxotrophic yeast strains in combination with vectors containing complementing prototrophic marker genes. Another approach uses heterologous genes or cassettes which, if present in the vector, render the otherwise sensitive yeast strain resistant to antibiotics. In addition, auto-selection systems for Saccharomyces cerevisiae have been developed that eliminate the requirement for synthetic drop-out media or the use of antibiotics for transformation selection and subsequent plasmid maintenance in expression cultures.

Trehalose as glucose surrogate in proliferation and cellular mobility of adult neural progenitor cells derived from mouse hippocampus.

The disaccharide trehalose (TRE) represents a natural energy supply for distinct non-mammalian species. Evidence has shown that TRE impacts on various properties including the stabilization of protein structure and cell membranes, which are important neuroprotective features against neurodegeneration. In this study, we tested the specific effect of TRE on cell proliferation and mobilization using an established experimental paradigm of adult neural progenitor cells (NPCs) derived from murine hippocampus.

Preloading budding yeast with all-in-one CRISPR/Cas9 vectors for easy and high-efficient genome editing.

The CRISPR/Cas9 technology has greatly improved genome editing in over recent years. However, several current CRISPR/Cas9 systems suffer from work-intensive cloning procedures and/or the requirement of co-transforming target cells with multiple system components simultaneously which can reduce the effectivity of such applications. Here, we present a new set of all-in-one CRISPR/Cas9 vectors that combine unique benefits of different already existent systems in order to further expand the technology's design possibilities.

Lipid determinants of endocytosis and exocytosis in budding yeast.

Cells maintain physicochemical characteristics of membranes in order to allow for proper function of membrane-associated cellular processes, such as endocytosis and exocytosis. To investigate the interplay between membrane properties and biological processes, we applied lipid engineering approaches that allowed for systematic manipulation of fatty acid unsaturation and sterol biosynthesis, the main regulators of membrane fluidity. In combination with electrophysiological membrane capacitance measurements, we were able to study the dependence of the endo- and exocytic activity of Saccharomyces cerevisiae on membrane lipid composition in vivo.

Trypanosoma brucei aquaglyceroporins mediate the transport of metabolic end-products: Methylglyoxal, D-lactate, L-lactate and acetate.

Bloodstream forms of Trypanosoma (T.) brucei, the causative agent of African sleeping sickness, possess a highly active glycolysis, which generates as main end-products: pyruvate under aerobic conditions, and pyruvate and glycerol under anaerobic conditions. To secrete them into the extracellular milieu, the parasites have at least two main specific membrane proteins, the pyruvate transporter and the aquaglyceroporins However, there are several other minor products from the glycolysis that must be excreted by the parasites and whose exit pathway until now remained elusive.

Vesicle fusion and fission in plants and yeast.

Measurements of the membrane capacitance on animal cells has provided an excellent technique for monitoring of exo- and endocytotic activity in intact living cells. Here we review recent data in which the same technique was applied to plant cells and cells of the budding yeast Saccharomyces cerevisiae. The data show that unitary exo- and endocytotic events can also be measured with the same technique after removing the cell wall from these cells.

Lipid engineering reveals regulatory roles for membrane fluidity in yeast flocculation and oxygen-limited growth.

Cells modulate lipid metabolism in order to maintain membrane homeostasis. Here we use a metabolic engineering approach to manipulate the stoichiometry of fatty acid unsaturation, a regulator of cell membrane fluidity, in Saccharomyces cerevisiae. Unexpectedly, reduced lipid unsaturation triggered cell-cell adhesion (flocculation), a phenomenon characteristic of industrial yeast but uncommon in laboratory strains.

Lost in traffic? The K(+) channel of lily pollen, LilKT1, is detected at the endomembranes inside yeast cells, tobacco leaves, and lily pollen.

Fertilization in plants relies on fast growth of pollen tubes through the style tissue toward the ovules. This polarized growth depends on influx of ions and water to increase the tube's volume. K(+) inward rectifying channels were detected in many pollen species, with one identified in Arabidopsis.

High-resolution membrane capacitance measurements for studying endocytosis and exocytosis in yeast.

Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell-attached configuration of the patch-clamp techniques we were able to resolve the elementary processes of endo- and exocytosis in yeast protoplasts at high temporal and spatial resolution.

Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K(+) channel, Tok1p.

Tandem pore-loop potassium channels differ from the majority of K(+) channels in that a single polypeptide chain carries two K(+)-specific segments (P) each sandwiched between two transmembrane helices (M) to form an MP(1)M-MP(2)M series. Two of these peptide molecules assemble to form one functional potassium channel, which is expected to have biaxial symmetry (commonly described as asymmetric) due to independent mutation in the two MPM units. The resulting intrinsic asymmetry is exaggerated in fungal 2P channels, especially in Tok1p of Saccharomyces, by the N-terminal presence of four more transmembrane helices.

Function of a separate NH3-pore in Aquaporin TIP2;2 from wheat.

Functional analysis of heterologously expressed TaTIP2;2 by means of stopped-flow spectrometric studies provide evidence for water and ammonia conductivity. A series of experiments under increasing pH indicate that the gaseous NH(3), rather than the ammonium ion NH(4)(+) was transported. Results from inhibitor studies strongly suggest that NH(3) is not transported in file with water, but through a separate pathway, which could be supplied by the 5th central pore in a tetramer conformation.

Characterization of plant aquaporins.

Plants have been reported to contain a large set of aquaporins (38 for Arabidopsis), which has been divided into four subfamilies on the basis of similarities in their amino acid sequences. They belong to the large superfamily of major intrinsic proteins (MIP), which was the basis for the nomenclature PIP, TIP, and NIP, also indicating the subcellular localization plasma membrane, tonoplast, and nodule of the respective founding member. The fourth subfamily of small and basic intrinsic proteins is not well characterized so far.

TPK1, a Ca(2+)-regulated Arabidopsis vacuole two-pore K(+) channel is activated by 14-3-3 proteins.

The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs).

Arbuscular mycorrhizal symbiosis and plant aquaporin expression.

Almost all land plants have developed a symbiosis with arbuscular mycorrhizal fungi. Establishment of the association is accompanied by structural changes in the plant root. During arbuscule formation fungal hyphae penetrate the root apoplast and install highly specialized interfaces for solute transport between plant and fungus.

In the yeast potassium channel, Tok1p, the external ring of aspartate residues modulates both gating and conductance.

The yeast plasma-membrane potassium channel, Tok1p, is a voltage-dependent outward rectifier, the gating and steady-state conductance of which are conspicuously modulated by extracellular [K(+)] ([K(+)](o)). Activation is slow at high [K(+)](o), showing time constants (tau(a)) of approximately 90 ms when [K(+)](o) is 150 mM (depolarizing step to +100 mV), and inactivation is weak (<30%) during sustained depolarization. Lowering [K(+)](o) accelerates activation, increases peak current, and enhances inactivation, so that at 15 mM [K(+)](o) tau(a) is less than 50 ms and inactivation suppresses approximately 60% of peak current.

TPK1 is a vacuolar ion channel different from the slow-vacuolar cation channel.

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K(+) channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca(2+)- and voltage-dependent outwardly rectifying plasma membrane K(+) channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca(2+) and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane.

Plant K(in) and K(out) channels: approaching the trait of opposite rectification by analyzing more than 250 KAT1-SKOR chimeras.

Members of the Shaker-like plant K(+) channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (K(in)) channels, or leak-like (K(weak)) channels, or depolarization-activated outward-rectifying (K(out)) channels. Here we created 256 chimeras between the K(in) channel KAT1 and the K(out) channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.

AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner.

The Arabidopsis tandem-pore K(+) (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen.

Electrophysiological analysis of the yeast V-type proton pump: variable coupling ratio and proton shunt.

Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase. Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and Pi present) or off (no ATP).