Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans

The dominant mutation deg-1(u38) results in a toxic gene product that leads to the late-onset degeneration of a small number of neurons in the nematode Caenorhabditis elegans. Both intragenic and extragenic mutations as well as changes in wild-type gene dosage can delay or block the time of onset of the neuronal deaths. The deg-1 gene has been cloned and a partial complementary DNA reveals that the gene encodes a novel protein that may act as a membrane receptor. Because the late-onset loss of specific sets of neurons, often as a result of dominant mutations, is characteristic of several human neurodegenerative diseases, the analysis of the deg-1 gene and its suppressors may provide a means of understanding the mechanisms underlying some of these human diseases.     

Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment

The pathways of intracellular traffic involved in antigen processing and presentation have been defined by immunoelectron microscopy. The export pathway for class II histocompatibility molecules and the antigen import pathway meet in a peripheral endocytic compartment having all the molecular machinery believed to be required for antigen processing and presentation, including internalized surface immunoglobulins, proteolytic enzymes and invariant chains. This compartment defines a site where peptides from endocytosed antigen can bind class II molecules en route to the cell surface for presentation to T cells.     

No evidence for illegitimate young in monogamous and polygynous warblers

In animals with internal fertilization, paternity is uncertain. In birds, the occurrence of copulations outside the pair-bond has been documented in a number of species, but the extent to which these result in illegitimate young is largely unknown, and constitutes a major deficiency in our understanding of avian mating systems. The analysis of tandemly repeated sequences (minisatellites), has enhanced our ability to make individual identifications and paternity determinations. Here we describe the use of a bird minisatellite DNA probe in assigning paternity in natural populations of the monogamous willow warbler Phylloscopus trochilus and of the polygynous wood warbler Phylloscopus sibilatrix. In both species this probe detects a multiple locus pattern and a single locus that exhibits a variable number of tandem repeats. Although we observed intrusions by non-resident males into the territories of paired males and extra-pair copulations, no illegitimate offspring were detected among 176 young from 32 families of both species, implying that extra-pair copulations have little or no genetic impact.     

Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein

The three-dimensional structure of the amino-terminal 44K ATPase fragment of the 70K bovine heat-shock cognate protein has been solved to a resolution of 2.2 A. The ATPase fragment has two structural lobes with a deep cleft between them; ATP binds at the base of the cleft. Surprisingly, the nucleotide-binding 'core' of the ATPase fragment has a tertiary structure similar to that of hexokinase, although the remainder of the structures of the two proteins are completely dissimilar, suggesting that both the phosphotransferase mechanism and the substrate-induced conformational change intrinsic to the hexokinases may be used by the 70K heat shock-related proteins.     

Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum

Complexes of cell-surface receptors and their ligands are commonly internalized by endocytosis and enter a prelysosomal endosomal pathway for further processing. Fluorescence microscopy and video recording of living cells to trace the passage of ligand-receptor complexes has identified the endosomal compartment as an extensive network of tubular cisternae. Endocytosed material entering this reticulum enters discrete swellings, identified as multivesicular bodies by electron microscopy, which move along the reticulum towards the pericentriolar area.     

Small G proteins are expressed ubiquitously in lymphoid cells and do not correspond to Bcl-2

The bcl-2 gene is consistently associated with t(14; 18) chromosomal translocations observed in a large fraction of human B-cell lymphomas. The t(14; 18) translocation results in deregulated expression of the bcl-2 gene and synthesis of inappropriately high levels of the Bcl-2 protein. Gene transfer studies suggest a role for Bcl-2 in cell survival, growth enhancement and oncogenic transformation. To test the suggestion that GTP-binding by Bcl-2 may mediate its biological effects we characterized the GTP-binding proteins in lymphoid cells expressing Bcl-2. Expression of several small GTP-binding proteins was found to be ubiquitous and did not vary with levels of Bcl-2. By using immunological, electrophoretic and cell-fractionation techniques, we separated Bcl-2 from G proteins of small relative molecular mass (Mr) and showed that it is incapable of binding GTP. Our results show that small Mr G proteins are widely expressed in lymphoid cells and that Bcl-2 is not a novel member of this GTP-binding protein family.     

Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.