Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms.

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.     

Molecular characterization of a blue-copper laccase, TILA, of Aspergillus nidulans

Laccases are blue-copper enzymes, which oxidize phenolic substrates and thereby reduce molecular oxygen. They are widespread within fungi and are involved in lignin degradation or secondary metabolism such as pigment biosynthesis. Many fungi contain several laccases, not all of whose functions are known. In Aspergillus nidulans one, yA, is expressed during asexual development and converts a yellow precursor to the green pigment. We identified a second laccase gene, which encodes a 66.3-kDa protein 37.6% identical to laccase I of A. nidulans. The protein harbors an N-terminal secretion signal, and three characteristic copper-binding centers. The enzyme localizes at the growing hyphal tip. The gene was therefore named tilA (=tip laccase). Deletion or overexpression of the gene had no discernible phenotype under laboratory conditions.     

Identification of specific pore residues mediating KCNQ1 inactivation. A novel mechanism for long QT syndrome

KCNQ1 inactivation bears electrophysiological characteristics different from classical N- and C-type inactivation in Shaker-like potassium channels. However, the molecular site of KCNQ1 inactivation has not yet been determined. KCNQ2 channels do not exert a fast inactivation in contrast to KCNQ1 channels. By expressing functional chimeras between KCNQ1 and KCNQ2 in Xenopus oocytes, we mapped the region of this inactivation to transmembrane domain S5 and the pore loop H5 and finally narrowed down the site to positions Gly(272) and Val(307) in KCNQ1. Exchanging these two amino acids individually with the analogous KCNQ2 residue abolished inactivation. Furthermore, a KCNQ1-like inactivation was introduced into KCNQ2 by mutagenesis in the corresponding region, confirming its relevance for the inactivation process. As KCNQ1 inactivation involves the regions S5 and H5, it exhibits a geography distinct from N- or C-type inactivation. Native cardiac I(Ks) channels comprising KCNQ1 and accessory MinK subunits do not inactivate because of the functional interaction of KCNQ1 with MinK. Mutations in KCNQ1 can lead to long QT1 syndrome, an inherited form of arrhythmia. The long QT1 mutant KCNQ1(L273F) displays a pronounced KCNQ1 inactivation. Here we show that when expressing mutant I(Ks) channels formed from KCNQ1(L273F) and MinK, MinK association no longer eliminates KCNQ1 inactivation. This results in smaller repolarizing currents in the heart and therefore represents a novel mechanism leading to long QT syndrome.     

Phospholipid-stimulated protein kinase in plants

In membrane fractions from zucchini (Cucurbita pepo L.) hypocotyls, catalytic properties of a platelet-activating factor (PAF)-activated protein kinase were investigated. In the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, phosphorylation of a 55-kDa membrane polypeptide and, to a lesser extent, several others, including a 120-kDa polypeptide, was stimulated by PAF. The phosphorylation of the 55-kDa polypeptide was used for quantification of the PAF-stimulated protein kinase. Stimulation of protein phosphorylation by PAF increased in a concentration range from 10-200 micrograms/ml (= 19-380 microM) PAF up to 10-fold above the control. Addition of Ca2+ ions in the micromolar range in the presence and in the absence of PAF increased the phosphorylation of the 55- and the 120-kDa polypeptide. Other phospholipids and lipids tested including phorbol ester, diglyceride, mono- and triglyceride, and oleic acid were ineffective. The same lipid specificity was previously observed for the activation of ATP-dependent H+ transport in microsomes (Scherer, G.F.E., Martiny-Baron, G., and Stoffel, B. (1988) Planta 175, 241-253). Lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) were able to stimulate the phosphorylation of the same polypeptides as PAF and H+ transport but both to a lesser extent (PAF greater than LPC greater than LPE). In the presence of EGTA, PAF-stimulated phosphorylation of a 55- and a 57-kDa polypeptide was predominantly associated with vacuolar membranes and those of 42, 61, 63, and 120 kDa were predominantly associated with plasma membranes. Stimulation of ATP-dependent H+ transport by PAF was found in tonoplast vesicles whereas plasma membrane vesicles had only little transport activity and, therefore, an effect of PAF on plasma membrane H+ transport could not be measured. Stimulation of ATP hydrolysis by PAF was observed both in tonoplast- and plasma membrane-containing fractions.     

Cellular localization of cytochrome c 553 in the N2-fixing cyanobacterium Anabaena variabilis

The in situ location of the electron carrier protein cytochrome C ₅₅₃ (cyt c ₅₅₃) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c ₅₅₃ specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c ₅₅₃ during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c ₅₅₃ is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c ₅₅₃ cytochrome c ₅₅₃ - PBS phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4) - PMSF phenylmethylsulfonyl fluoride Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz.