Identification of 2n breeding lines and 4n varieties of potato (Solanum tuberosum,ssp. tuberosum) with RFLP-fingerprints

Summary The possibility of genotype identification with RFLP fingerprints was examined with 20 tetraploid potato varieties and 38 diploid potato lines. By using a sensitive detection system for small restriction fragment length differences and highly variable potato sequences as probes, all genotypes (diploids and tetraploids) were distinguished by a minimum of two probe/enzyme combinations. The best single probe/enzyme combination distinguished 19 out of 20 4n varieties and 33 out of 38 2n lines. Intravarietal variability was very small compared to the intervarietal variability, and patterns obtained with different DNA sources of the same genotype were identical.     

Analysis of interaction partners of H4 histone by a new proteomics approach

We describe a modification of the tandem affinity purification method for purification and analysis of multiprotein complexes, termed here DEF‐TAP (for differential elution fractionation after tandem affinity purification). Its essential new feature is the use for last purification step of 6×His‐Ni⁺⁺ interaction, which is resistant to a variety of harsh washing conditions, including high ionic strength and the presence of organic solvents. This allows us to use various fractionation schemes before the protease digestion, which is expected to improve the coverage of the analyzed protein mixture and also to provide an additional insight into the structure of the purified macromolecular complex and the nature of protein–protein interactions involved. We illustrate our new approach by analysis of soluble nuclear complexes containing histone H4 purified from HeLa cells. In particular, we observed different fractionation patterns of HAT1 and RbAp46 proteins as compared with RbAp48 protein, all identified as interaction partners of H4 histone. In addition, we report all components of the licensing MCM2‐7 complex and the apoptosis‐related DAXX protein among the interaction partners of the soluble H4. Finally, we show that HAT1 requires N‐terminal tail of H4 for its stable association with this histone.     

Hepatitis B virus HBx peptide 116-138 and proteasome activator PA28 compete for binding to the proteasome alpha4/MC6 subunit

PA28 is a modulator of the 20S proteasome. The PA28 binding sites on the 20S proteasome are still not well defined. Using yeast two-hybrid interaction assays and proteasome inactivation kinetics we provide evidence that the proteasome alpha4 subunit is one of the PA28 binding sites. This finding is supported by the observation that a hepatitis B virus X protein-derived polypeptide habouring the alpha4 proteasome subunit binding motif impairs the activation of 20S proteasomes by PA28.     

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells

Summary Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture of flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry.A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanom a cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modified by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10^–6 oligomycin.When atebrine at levels (10^–6 M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g. 2×10^–5 M) an irreversible increase of atebrine fluorescence is seen.The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.     

Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus equisimilis

Catalytic and regulatory domains of the Rel/Spo homolog of Streptococcus equisimilis affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared. Two major domains of the 739-residue Rel(Seq) protein are defined by limited proteolytic digestion. In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3'-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn(2+)-dependent 3'-pyrophosphohydrolase. Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein. Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis. Cellular (p)ppGpp accumulation behavior is consistent with these changes. The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro. These assignments can also apply to RelA and SpoT. The ability of Rel(Seq) to mediate (p)ppGpp accumulation during amino acid starvation in S. equisimilis is absent when the protein is expressed ectopically in Escherichia coli. Fusing the N-terminal half of Rel(Seq) with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in E. coli by inhibiting unregulated degradation and restoring regulated synthetic activity. Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins.     

Identification of a Probable Pore-Forming Domain in the Multimeric Vacuolar Anion Channel AtALMT9

Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an “open channel blocker” of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.The transport of ions across cellular membranes is mediated by specialized proteins that catalyze the transfer of charged molecules across hydrophobic lipid bilayers. Based on the thermodynamics, two major classes of transport systems can be distinguished: (1) passive transporters such as ion channels, which catalyze the flux of solutes down the electrochemical gradient, and (2) active transporters like pumps and antiporters, which transport molecules against their electrochemical gradient. Independent of the nature of the transport system, the flux of ions across membranes is crucial for a wide range of physiological functions in plants. Among others, ion transport is involved in intracellular pH regulation, metal tolerance, stomatal movement, cellular signaling, plant nutrition, and cell expansion (Roelfsema and Hedrich, 2005; Kim et al., 2010; Barbier-Brygoo et al., 2011). Despite the importance of anion transport in plant physiology, only in the last decade has the molecular identity of anion transport proteins started to be unveiled by identifying the chloride channel (CLC), slow anion channel (SLAC), and aluminum-activated malate transporter (ALMT) families. Their discovery has been a fundamental breakthrough in understanding the molecular mechanisms of anion homeostasis and its roles in various aspects of plant cell physiology (Ward et al., 2009; Barbier-Brygoo et al., 2011; Hedrich, 2012; Martinoia et al., 2012).The CLC family consists of both anion channels and secondary active transporters, which are ubiquitously expressed in all living organisms. In Arabidopsis (Arabidopsis thaliana), the first identified and characterized member of the family was AtCLCa (Hechenberger et al., 1996; Geelen et al., 2000). AtCLCa is targeted to the tonoplast and acts as a 2NO₃⁻/H⁺ antiporter (De Angeli et al., 2006). In planta, AtCLCa represents a major vacuolar nitrate transporter driving the accumulation of this anion into the vacuole. Subsequent studies revealed that all other Arabidopsis CLCs are likewise localized in intracellular membranes but feature different cellular functions (Barbier-Brygoo et al., 2011).The SLAC protein family was identified in the last decade (Negi et al., 2008; Vahisalu et al., 2008). Despite its recent discovery, the characterization of this plant anion transporter family proceeded rapidly (Negi et al., 2008; Vahisalu et al., 2008; Geiger et al., 2009, 2010; Brandt et al., 2012). SLAC1, the first identified member of the family, is involved in slow-type anion currents across the plasma membrane of plant cells (Negi et al., 2008; Vahisalu et al., 2008). This ion channel is expressed in guard cells, where it mediates the efflux of anions into the apoplast, a process that is fundamental for stomata closure. SLAC1 regulates the stomatal aperture in response to different stimuli such as abscisic acid and high CO₂ and ozone concentrations (Negi et al., 2008; Vahisalu et al., 2008). In addition, the activity of SLAC1 is controlled by different kinases (Geiger et al., 2009, 2010) that are part of various signaling pathways. This multiple regulation of SLAC1 suggests that it plays a critical role in the integration of different environmental stimuli.ALMTs are membrane proteins exclusive to plants. In Arabidopsis, this family consists of 14 members that can be grouped into three clades (Kovermann et al., 2007). The first member of the ALMT family, TaALMT1, was identified in wheat (Triticum aestivum) by Sasaki et al. (2004) when screening for genes associated with aluminum resistance. They provided evidence that TaALMT1 as well as AtALMT1, its homolog in Arabidopsis, are channels that catalyze the efflux of malate across the plasma membrane of root cells (Yamaguchi et al., 2005; Hoekenga et al., 2006). This exudation of organic acids into the soil facilitates the detoxification of environmental Al³⁺. Besides contributing to Al³⁺ tolerance, ALMTs have been found to exhibit other important physiological functions. AtALMT12 has been proposed to mediate rapid anion currents across the plasma membrane of guard cells in order to induce stomata closure (Meyer et al., 2010). AtALMT9 and AtALMT6 have been shown to be channels localized in the tonoplast that mediate the export of malate into the vacuole (Kovermann et al., 2007; Meyer et al., 2011). AtALMT6 is predominantly expressed in guard cells, where its activity is regulated by cytosolic Ca²⁺ and vacuolar pH (Meyer et al., 2011). In contrast, AtALMT9 is widely expressed in several plant tissues, such as the mesophyll and guard cells. Recently, AtALMT9 was shown to play a crucial role in stomata movement, where it functions as a malate-activated chloride channel (De Angeli et al., 2013).The knowledge about ion channel structures has expanded considerably in the last 20 years. Notably, various three-dimensional structures of such proteins have been solved (Choe, 2002; Jentsch, 2008; Traynelis et al., 2010). This has boosted the research into and the understanding of structure-function relations in transport systems. Among the anion channel families described above, the structure has been determined for CLCs (Dutzler et al., 2002) and SLACs (Chen et al., 2010). Additionally, large structure-function analyses have been conducted, providing detailed knowledge on the molecular basis underlying the ion channel functionality of these families. In contrast, little information was revealed about the structure of ALMTs by describing an important phosphorylation site (Ligaba et al., 2009; Furuichi et al., 2010) and by providing data on the topology (Motoda et al., 2007; Dreyer et al., 2012). However, the proposed models in these studies do not entirely coincide regarding the number of transmembrane-spanning domains, the cellular orientation of the N terminus, and the organization of the C-terminal domain. Therefore, the structural organization of ALMTs is still ambiguous.In this study, we performed a large-scale mutagenesis analysis of the transmembrane domain (TMD) of Arabidopsis ALMTs using the vacuolar channel AtALMT9 as a model. The aim was to identify regions of the TMD that potentially exhibit functional relevance by forming the pore or the voltage sensor. For that purpose, we took advantage of citrate, which we identified as an open channel blocker of AtALMT9. The use of this blocker allowed elucidation of the structural details of ion channels, such as the quaternary organization and pore-forming domains, when no crystal structure was available (MacKinnon, 1991; Yellen et al., 1991; Ferrer-Montiel and Montal, 1996; Linsdell, 2005). By this means, it is possible to show, for instance, that potassium channels are tetramers and to identify their “selectivity filter” domain (MacKinnon and Yellen, 1990; MacKinnon, 1991). Thus, by using citrate, we pharmacologically investigated structure-function relations in AtALMT9. We identified a region adjacent to and within the fifth putative TMD that is supposedly involved in forming the permeation pathway of AtALMT9. Moreover, we demonstrated that AtALMT9 is a multimeric channel of probably four subunits in which the monomers participate in forming the pore.     

Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers - effects beyond electrostatic

The presence of charged lipids in the cell membrane constitutes the background for the interaction with numerous membrane proteins. As a result, the valence of the lipids plays an important role concerning their lateral organization in the membrane and therefore the very manner of this interaction. This present study examines this aspect, particularly regarding to the interaction of the anionic lipid DPPS with the highly basic charged effector domain of the MARCKS protein, examined in monolayer model systems. Film balance, fluorescence microscopy and X-ray reflection/diffraction measurements were used to study the behavior of DPPS in a mixture with DPPC for its dependance on the presence of MARCKS (151-175). In the mixed monolayer, both lipids are completely miscible therefore DPPS is incorporated in the ordered crystalline DPPC domains as well. The interaction of MARCKS peptide with the mixed monolayer leads to the formation of lipid/peptide clusters causing an elongation of the serine group of the DPPS up to 7? in direction to surface normal into the subphase. The large cationic charge of the peptide pulls out the serine group of the interface which simultaneously causes an elongation of the phosphodiester group of the lipid fraction too. The obtained results were used to compare the interaction of MARCKS peptide with the polyvalent PIP(2) in mixed monolayers. On this way we surprisingly find out, that the relative small charge difference of the anionic lipids causes a significant different interaction with MARCKS (151-175). The lateral arrangement of the anionic lipids depends on their charge values and determines the diffusion of the electrostatic binding clusters within the membrane.