RESUMO
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. Baker's yeast, which was successfully employed for testing ion channels from eukaryotes has not yet been used for screening of ChR2s, because they neither produce the retinal chromophore nor its precursor carotenoids. We found that addition of retinal to the external medium was not sufficient for detecting robust ChR activity in yeast in simple growth assays. This obstacle was overcome by metabolic engineering of a yeast strain, which constitutively produces retinal. In proof of concept experiments we functionally express different ChR variants in these cells and monitor their blue light induced activity in simple growth assays. We find that light activation of ChR augments an influx of Na+ with a consequent inhibition of cell growth. In a K+ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K+ conductance of ChR. This yeast strain can now be used as chassis for screening of new functional ChR variants and mutant libraries in simple yeast growth assays under defined selective conditions.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Engenharia Metabólica , Mutação , FermentaçãoRESUMO
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. Here we describe a combination of host strain and vector system introducing a novel concept of auto-selection systems that allows for easy and robust propagation of host cells deleted in essential genes in supplemented media before being transformed with rescuing plasmids. With that, our approach is favorable over commonly used selection strategies and has major advantage over other auto-selection systems. Our approach complements the auto-selection toolbox already available for S. cerevisiae, thus contributing a novel system that enables the use of complex peptone-based media for protein expression and metabolic engineering approaches. We therefore expect that this new strategy will be of general interest to the yeast research community in academia and industry.
Assuntos
Engenharia Metabólica , Saccharomyces cerevisiae , Antibacterianos , Meios de Cultura , Vetores Genéticos , Plasmídeos/genética , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
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. We found that a strong decrease in the cell's total ergosterol content leads to a severely reduced frequency of vesicle fission (endocytosis), whereas the exocytic activity remained largely unaffected. In contrast, increased lipid saturation lowered both endocytic and the exocytic activity, with the former being more severely affected. We were able to correlate the decreased ratio of endocytic/exocytic frequencies (fendo/fexo) upon lipid perturbation with the growth of yeast protoplasts, which is based on a surface enlargement resulting from a net excess of exocytic over endocytic flux. Experiments using clathrin-deficient mutants confirm a correlation between reduced endocytic activity and increased size of intact walled cells, as well as accelerated protoplast growth. These data show that lipid composition is intimately tied to membrane trafficking in yeast cells and suggest that endocytosis is particularly dependent on the lipid-defined properties of cell membrane.
Assuntos
Endocitose , Exocitose , Fluidez de Membrana/fisiologia , Saccharomycetales/fisiologia , Crescimento Celular , Ergosterol/farmacologia , Lipídeos de Membrana/metabolismo , ProtoplastosRESUMO
By vacuolar patch-clamp and Ca2+ imaging experiments, we show that the yeast vacuolar transient receptor potential (TRPY) channel 1 is activated by cytosolic Ca2+ and inhibited by Ca2+ from the vacuolar lumen. The channel is cooperatively affected by vacuolar Ca2+ (Hill coefficient, 1.5), suggesting that it may accommodate a Ca2+ receptor that can bind two calcium ions. Alanine scanning of six negatively charged amino acid residues in the transmembrane S5 and S6 linker, facing the vacuolar lumen, revealed that two aspartate residues, 401 and 405, are essential for current inhibition and direct binding of 45Ca2+. Expressed in HEK-293 cells, a significant fraction of TRPY1, present in the plasma membrane, retained its Ca2+ sensitivity. Based on these data and on homology with TRPV channels, we conclude that D401 and D405 are key residues within the vacuolar vestibule of the TRPY1 pore that decrease cation access or permeation after Ca2+ binding.
RESUMO
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. As aquaglyceroporins from T. brucei (TbAQP1, 2, and 3) show a wide permeability profile for small solutes, we decided to evaluate if these proteins allow the passage of methylglyoxal, L-lactate, D-lactate and acetate molecules. We expressed heterologously TbAQP1, 2, and 3 in aquaglyceroporin-null yeast cells or in Xenopus laevis oocytes and demonstrated that these channels are permeable for methylglyoxal, L-lactate, D-lactate and acetate. We further demonstrate that methylglyoxal is highly toxic for bloodstream forms of T. brucei, while L-lactate and D-lactate appear almost harmless. Additionally, we discuss all our findings in the light of the novel metabolic discoveries, putting in context the participation of TbAQP1, 2, 3, and other proteins in the excretion of unwanted metabolic end-products.
Assuntos
Acetatos/metabolismo , Aquagliceroporinas/metabolismo , Ácido Láctico/metabolismo , Aldeído Pirúvico/metabolismo , Trypanosoma brucei brucei/metabolismo , Transporte Biológico , Glicerol/metabolismo , Glicólise , Ácido Pirúvico/metabolismo , EstereoisomerismoRESUMO
The CRISPR/Cas9 technology has greatly improved genome editing in Saccharomyces cerevisiae 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. Our vectors mediate constitutive gRNA expression whereas Cas9 expression is either driven from a constitutive or an inducible promoter. The introduction of desired gRNA targeting sequences into our inducible single gRNA vector relies just on in vivo homologous recombination-mediated assembly of overlapping single-stranded oligonucleotides, thus reducing efforts of plasmid cloning to an absolute minimum. By employing the inducible system, yeast cells can be easily preloaded with plasmids encoding for a functional CRISPR/Cas9 system, thereby chronologically separating the cloning procedure from the genome editing step. Gene knockouts could be achieved with high efficiency and effectivity by simply transforming preloaded cells with a selectable disruption cassette without the need of co-introducing any CRISPR/Cas9 system component. We also show the feasibility of efficient gene knockouts even when multiple gene copies were present such as in non-haploid strain backgrounds as well as the simultaneous deletion of two different genes in a haploid genetic background by using a multiplex variant of our inducible vector. The versatile applicability of our inducible vector system was further demonstrated by CRISPR/Cas9-mediated mating type switching of yeast.
RESUMO
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. The resulting protoplasts execute the same type of transient and permanent fusion/fission that is known from animal cells. Also the size of the vesicles, which are fusing or budding, are of the same order of magnitude as those recorded in animal cells. Together these data support the view of an evolutionary conserved mechanism for unitary exo- and endocytosis events in eukaryotes. The successful recordings of exo- and endocytotic activity in Saccharomyces cerevisiae by capacitance measurements now pave the way for correlating the abundant information on the molecular machinery of exo- and endocytosis in this model organism with distinct functional properties.
Assuntos
Cálcio/metabolismo , Fusão de Membrana , Potenciais da Membrana/fisiologia , Protoplastos/metabolismo , Saccharomyces cerevisiae/metabolismo , Zea mays/metabolismo , Transporte Biológico , Membrana Celular , Cotilédone/citologia , Cotilédone/metabolismo , Capacitância Elétrica , Endocitose/fisiologia , Exocitose/fisiologia , Técnicas de Patch-Clamp , Protoplastos/ultraestrutura , Saccharomyces cerevisiae/citologia , Zea mays/citologiaRESUMO
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. We find that ER lipid saturation sensors induce expression of FLO1 - encoding a cell wall polysaccharide binding protein - independently of its canonical regulator. In wild-type cells, Flo1p-dependent flocculation occurs under oxygen-limited growth, which reduces unsaturated lipid synthesis and thus serves as the environmental trigger for flocculation. Transcriptional analysis shows that FLO1 is one of the most highly induced genes in response to changes in lipid unsaturation, and that the set of membrane fluidity-sensitive genes is globally activated as part of the cell's long-term response to hypoxia during fermentation. Our results show how the lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to an environmental stimulus important in biotechnology.
Assuntos
Membrana Celular , Engenharia Genética , Lectinas de Ligação a Manose , Fluidez de Membrana/genética , Lipídeos de Membrana , Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Celular/genética , Membrana Celular/metabolismo , Floculação , Lectinas de Ligação a Manose/biossíntese , Lectinas de Ligação a Manose/genética , Lipídeos de Membrana/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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. Spontaneous capacitance changes were predominantly in the range of 0.2-1 fF which translates to vesicle diameters of 90-200 nm. The size distribution revealed that endocytotic vesicles with a median at about 132 nm were smaller than exocytotic vesicles with a median at 155 nm. In energized and metabolizing protoplasts, endo- and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts.
Assuntos
Membrana Celular/metabolismo , Endocitose , Exocitose , Potenciais da Membrana , Saccharomyces cerevisiae/metabolismo , Membrana Celular/fisiologiaRESUMO
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. Here, an Arabidopsis AKT1-like channel (LilKT1) was identified from Lilium longiflorum pollen. Complementation of K(+) uptake deficient yeast mutants was only successful when the entire LilKT1 C-terminus was replaced by the AKT1 C-terminus. No signals were observed in the plasma membrane (PM) of pollen tubes after expression of fluorescence-tagged LilKT1 nor were any LilKT1-derived peptides detectable in the pollen PM by mass spectrometry analysis. In contrast, fluorescent LilKT1 partly co-localized with the lily PM H(+) ATPase LilHA2 in the PM of tobacco leaf cells, but exhibited a punctual fluorescence pattern and also sub-plasma membrane localization. Thus, incorporation of LilKT1 into the pollen PM seems tighter controlled than in other cells with still unknown trafficking signals in LilKT1's C-terminus, resulting in channel densities below detection limits. This highly controlled incorporation might have physiological reasons: an uncontrolled number of K(+) inward channels in the pollen PM will give an increased water influx due to the raising cytosolic K(+) concentration, and finally, causing the tube to burst.
RESUMO
Salinity tolerance in plants involves controlled Na(+) transport at the site of Na(+) accumulation and intracellular Na(+) compartmentation. The focus of this study was the identification and analysis of the expression of Na(+)/H(+) antiporters in response to NaCl stress in one particular plant, the facultative halophyte Mesembryanthemum crystallinum Na(+)/H(+) antiporters of M. crystallinum were cloned by RACE-PCR from total mRNA of leaf mesophyll cells. Functional complementation of Saccharomyces cerevisiae and Escherichia coli mutants was performed. The kinetics of changes in the expression of antiporters were quantified by real-time PCR in leaves and roots. Five Na(+)/H(+) antiporters (McSOS1, McNhaD, McNHX1, McNHX2 and McNHX3) were cloned, representing the entire set of these transporters in M. crystallinum. The functionality of McSOS1, McHX1 and McNhaD was demonstrated in complementation experiments. Quantitative analysis revealed a temporal correlation between salt accumulation and expression levels of genes in leaves, but not in roots, which was most pronounced for McNhaD. Results suggest a physiological role of McSOS1, McNhaD and McNHX1 in Na(+) compartmentation during plant adaptation to high salinity. The study also provides evidence for salt-induced expression and function of the Na(+)/H(+) antiporter McNhaD in chloroplasts and demonstrates that the chloroplast is one of the compartments involved in the response of cells to salt stress.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mesembryanthemum/genética , Folhas de Planta/genética , Raízes de Plantas/genética , Cloreto de Sódio/farmacologia , Trocadores de Sódio-Hidrogênio/genética , Estresse Fisiológico/genética , Cloroplastos/efeitos dos fármacos , Cloroplastos/genética , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Perfilação da Expressão Gênica , Teste de Complementação Genética , Proteínas de Fluorescência Verde/metabolismo , Higromicina B/farmacologia , Mutação/genética , Filogenia , Folhas de Planta/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Fatores de TempoRESUMO
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. Functional implications of such structural asymmetry have been investigated via mutagenesis of residues (L290 in P(1) and Y424 in P(2)) that are believed to provide the outermost ring of carbonyl oxygen atoms for coordination with potassium ions. Both complementary mutations (L290Y and Y424L) yield functional potassium channels having quasi-normal conductance when expressed in Saccharomyces itself, but the P(1) mutation (only) accelerates channel opening about threefold in response to depolarizing voltage shifts. The more pronounced effect at P(1) than at P(2) appears paradoxical in relation to evolution, because a comparison of fungal Tok1p sequences (from 28 ascomycetes) shows the filter sequence of P(2) (overwhelmingly TIGYGD) to be much stabler than that of P(1) (mostly TIGLGD). Profound functional asymmetry is revealed by the fact that combining mutations (L290Y + Y424L)-which inverts the order of residues from the wild-type channel-reduces the expressed channel conductance by a large factor (20-fold, cf. Assuntos
Sequência de Aminoácidos
, Leucina/genética
, Canais de Potássio/genética
, Proteínas de Saccharomyces cerevisiae/genética
, Tirosina/genética
, Teste de Complementação Genética
, Dados de Sequência Molecular
, Mutação
, Técnicas de Patch-Clamp
, Filogenia
, Canais de Potássio/classificação
, Canais de Potássio/metabolismo
, Estrutura Secundária de Proteína
, Proteínas de Saccharomyces cerevisiae/classificação
, Proteínas de Saccharomyces cerevisiae/metabolismo
, Alinhamento de Sequência
RESUMO
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.
Assuntos
Aquaporinas/metabolismo , Compostos de Amônio Quaternário/metabolismo , Triticum/metabolismo , Aquaporinas/genética , Transporte Biológico , Concentração de Íons de Hidrogênio , Triticum/genética , Água/metabolismoRESUMO
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. The increasing number of reports dealing with various aspects of plant aquaporins is starting to advance our understanding of aquaporin biology in plants. Fundamental questions include: what is the basic function of the different plant aquaporins, what is their primary substrate, and what is the consequence of function/malfunction of a particular aquaporin for the overall function of the plant? Biochemical and biophysical techniques can be employed to get information on the basic functional characteristics of plant aquaporins. An impressive set of techniques has been used to study aquaporin function on molecular, subcellular, and cellular levels in plants, as well as in heterologous expression systems. The physiological role of aquaporins in plants is much less well understood, but reports unraveling the physiological role of aquaporins, mainly employing genetic techniques and functional measurement on the whole plant level, are emerging. The goal of this chapter is to give an overview on the applied methods, together with some exemplary findings.
Assuntos
Aquaporinas/fisiologia , Fenômenos Fisiológicos Vegetais , Animais , Membrana Celular/fisiologia , Tamanho Celular , Clorofila/química , Concentração de Íons de Hidrogênio , Oócitos/fisiologia , Pressão Osmótica , Permeabilidade , Fotossíntese/fisiologia , Plantas/genética , Interferência de RNA , Saccharomyces cerevisiae/fisiologia , Xenopus laevisRESUMO
Monoclonal antibodies against the K(+) channel KAT1 of Arabidopsis thaliana, a low abundance, plant plasma membrane protein, were generated by genetic immunisation to avoid the time and labour consuming purification of native or recombinant proteins and peptides usually necessary for conventional immunisation techniques. The resulting polyclonal and monoclonal antibody sera recognised a single protein band in a microsomal fraction of wild-type A. thaliana leaves and in membrane fractions of transgenic yeast cells and tobacco plants expressing the KAT1 protein. Therefore, genetic immunisation is suitable for generating monoclonal antibodies against plant proteins and particularly, against plant membrane proteins of low abundance.
Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/imunologia , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Feminino , Vetores Genéticos , Hibridomas/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Plasmídeos/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Saccharomyces cerevisiae/genética , Nicotiana/genética , Vacinas de DNA/genéticaRESUMO
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. The periarbuscular membrane which is part of the plant plasma membrane surrounding arbuscular structures was shown to harbour a high density of different transport systems. Among these also expression of aquaporins was described, which potentially can act as a low affinity transport system for ammonia or ammonium. The present study provides data for expression, localization and function of plant aquaporins in the periarbuscular membrane of mycorrhizal Medicago truncatula plants.
Assuntos
Aquaporinas/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Micorrizas/metabolismo , Simbiose/fisiologia , Aquaporinas/genética , Micorrizas/químicaRESUMO
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. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mM KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca(2+), requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K(+) over Na(+), and its activity turned out to be independent of the membrane voltage over the range of +/-80 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca(2+)-activated, K(+)-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Sinalização do Cálcio , Condutividade Elétrica , Expressão Gênica , Membranas Intracelulares/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genéticaRESUMO
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.e., which are functionally equivalent to KAT1. This strategy allowed us to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N-terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the K(out) channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-I314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Potássio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Arabidopsis/genética , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Canais de Potássio/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Superfamília Shaker de Canais de Potássio , Relação Estrutura-AtividadeRESUMO
SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K(+) channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K(out) channels. Deletion mutants and chimeric proteins generated from SKOR and the K(in) channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K(T) domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K(out) alpha-subunits did not assemble with K(in) alpha-subunits because of the absence of interaction between their assembly sites.