AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.     

Current–voltage–time records of ion translocating enzymes

Membrane currents, as non-linear functions of membrane voltage, V, and time, t, can be recorded quickly by triangular V protocols. From the differences, dI(V,t), of these relationships upon addition of a putative substrate of a charge-translocating membrane protein, the I(V,t) relationships of the transporter itself can be determined. These relationships likely comprise a steady-state component, I_a(V), of the active transporter, and a dynamic component, p_a(V,t), of its V- and time-dependent activity, p_a. Here, the steady-state component is modeled by a central reaction cycle, which senses a fraction _tr of the total V, whereas 1–_tr can be assigned to an inner and outer pore section with _i and _o, respectively (_i+_tr+_o = 1). For the enzymatic cycle, fast binding/debinding is assumed, plus V-sensitive and -insensitive reaction steps which may become rate limiting for charge translocation. At given substrate concentrations, I_a(V) is defined by eight independent system parameters, including a coefficient for the barrier shape of charge translocation. In ordinary cases, the behavior of p_a(V,t) can be described by two rate constants (for activation and inactivation) and their respective V-sensitivity coefficients. Here, the effects of the individual system parameters on I(V,t) from triangular V-clamp experiments are investigated systematically. The results are illustrated by panels of typical curve shapes for non-gated and gated transporters to enable a first classification of mechanisms. We demonstrate that all system parameters can be determined fairly well by fitting the model to experimental data of known origin. Applicability of the model to channels, pumps and cotransporters is discussed.     

The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion

The NSF homolog Sec18 initiates fusion of yeast vacuoles by disassembling cis-SNARE complexes during priming. Sec18 is also required for palmitoylation of the fusion factor Vac8, although the acylation machinery has not been identified. Here we show that the SNARE Ykt6 mediates Vac8 palmitoylation and acts during a novel subreaction of vacuole fusion. This subreaction is controlled by a Sec17-independent function of Sec18. Our data indicate that Ykt6 presents Pal-CoA via its N-terminal longin domain to Vac8, while transfer to Vac8's SH4 domain occurs spontaneously and not enzymatically. The conservation of Ykt6 and its localization to several organelles suggest that its acyltransferase activity may also be required in other intracellular fusion events.     

Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.     

Convergent evolution of chromosomal sex-determining regions in the animal and fungal kingdoms

Sexual identity is governed by sex chromosomes in plants and animals, and by mating type (MAT) loci in fungi. Comparative analysis of the MAT locus from a species cluster of the human fungal pathogen Cryptococcus revealed sequential evolutionary events that fashioned this large, highly unusual region. We hypothesize that MAT evolved via four main steps, beginning with acquisition of genes into two unlinked sex-determining regions, forming independent gene clusters that then fused via chromosomal translocation. A transitional tripolar intermediate state then converted to a bipolar system via gene conversion or recombination between the linked and unlinked sex-determining regions. MAT was subsequently subjected to intra- and interallelic gene conversion and inversions that suppress recombination. These events resemble those that shaped mammalian sex chromosomes, illustrating convergent evolution in sex-determining structures in the animal and fungal kingdoms.     

Recombination: Holliday junction resolution and crossover formation

The heterodimeric nuclease Mus81-Eme1 has been proposed to be a Holliday junction resolvase and has now been found to be responsible for nearly all meiotic crossovers in fission yeast. The intriguing substrate preference of this enzyme for nicked Holliday junctions opens the possibility that crossover formation may not always involve double Holliday junctions.     

Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation

Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase–loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3′-phosphotyrosine protein–DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre–loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a ‘cleavage-competent’ Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3′-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the ‘cleavage-competent’ subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the ‘non-cleaving’ subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer.     

Long-term results and learning curve for radio frequency ablation of accessory pathways

Radio frequency (RF) catheter ablation of accessory pathways represents an interventional method in modern cardiology that has become the first-line treatment for patients with symptomatic WPW-syndrome. The aim of this study was to analyze: (1) the learning curve for the ablation procedure; (2) procedural parameters and success; and (3) personal assessment of the treatment by the patients. Learning curve analysis included 195 consecutive patients, who underwent ablation between 1991 and 1996. The follow-up survey included 65 consecutive patients. The analysis of the procedural parameters showed significant improvement after 100 cases, implying a completion of the learning curve at this point. Long-term follow-up showed a high success rate for all pathways (95.4%). All procedure parameters indicated significantly higher degree of difficulty for right free-wall and septal pathways, with lowest long-term success rate for right-sided pathways (78.6%). Personal assessment survey showed high acceptance of the treatment; the procedure was described as a significant improvement of overall quality-of-life by 92.3% of patients. The results of this study confirm the catheter ablation of accessory pathways--in particular after completion of the learning curve--as a low-risk and highly efficient treatment for symptomatic WPW-syndrome, with a high degree of patient-related acceptance.     

Holliday junctions in the eukaryotic nucleus: resolution in sight?

The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.