An essential role for a phospholipid transfer protein in yeast Golgi function

Progression of proteins through the secretory pathway of eukaryotic cells involves a continuous rearrangement of macromolecular structures made up of proteins and phospholipids. The protein SEC14p is essential for transport of proteins from the yeast Golgi complex. Independent characterization of the SEC14 gene and the PIT1 gene, which encodes a phosphatidylinositol/phosphatidylcholine transfer protein in yeast, indicated that these two genes are identical. Phospholipid transfer proteins are a class of cytosolic proteins that are ubiquitous among eukaryotic cells and are distinguished by their ability to catalyse the exchange of phospholipids between membranes in vitro. We show here that the SEC14 and PIT1 genes are indeed identical and that the growth phenotype of a sec14-1ts mutant extends to the inability of its transfer protein to effect phospholipid transfer in vitro. These results therefore establish for the first time an in vivo function for a phospholipid transfer protein, namely a role in the compartment-specific stimulation of protein secretion.     

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.     

Transparency and coherence in human motion perception

When confronted with moving images, the visual system often must decide whether the motion signals arise from a single object or from multiple objects. A special case of this problem arises when two independently moving gratings are superimposed. The gratings tend to cohere and move unambiguously in a single direction (pattern motion) instead of moving independently (component motion). Here we report that the tendency to see pattern motion depends very strongly on the luminance of the intersections (that is, to regions where the gratings overlap) relative to that of the gratings in a way that closely parallels the physics of transparency. When the luminance of these regions is chosen appropriately, pattern motion is destroyed and replaced by the appearance of two transparent gratings moving independently. The observations imply that motion detecting mechanisms in the visual system must have access to tacit 'knowledge' of the physics of transparency and that this knowledge can be used to segment the scene into different objects. The same knowledge could, in principle, be used to avoid confusing shadows with real object boundaries.     

Cryopreservation of Drosophila melanogaster embryos

There is an urgent need to preserve the ever-increasing number (greater than 30,000) of different genetic strains of D. melanogaster that are maintained in national and international stock centres and in the laboratories of individual investigators. In all cases, the stocks are maintained as adult populations and require transfer to fresh medium every two to four weeks. This is not only costly in terms of materials, labour and space, but unique strains are vulnerable to accidental loss, contamination, and changes in genotype that can occur during continuous culture through mutation, genetic drift or selection. Although cryopreservation of Drosophila germ-plasm would be an enormous advantage, many attempts using conventional procedures have been unsuccessful. D. melanogaster embryos are refractory to conventional cryopreservation procedures because of the contravening conditions required to minimize mortality resulting from both intracellular ice formation and chilling injury at subzero temperatures. To overcome these obstacles, we have developed a vitrification procedure that precludes intracellular ice formation so that the embryos can be cooled and warmed at ultra-rapid rates to minimize chilling injury, and have recovered viable embryos following storage in liquid nitrogen. In a series of 53 experiments, a total of 3,711 larvae emerged from 17,280 eggs that were cooled in liquid nitrogen (18.4 +/- 8.8%). Further, using a subset from this population, approximately 3% of the surviving larvae (24/800) developed into adults. These adults were fertile and produced an F1 generation.     

Immune sera recognize on erythrocytes Plasmodium falciparum antigen composed of repeated amino acid sequences

Protective immune responses against the asexual stages of the human malaria parasite, Plasmodium falciparum, are most probably directed against exposed antigenic determinants on the surface of the free merozoite or the infected red blood cell, and therefore antigens in these locations are candidates for testing as components of a defined molecular vaccine. To facilitate the search for such antigens, we recently developed a method for the expression of P. falciparum proteins in Escherichia coli as fused polypeptides. Many clones producing antigens were detected by screening with immune human sera. We show here that antibodies against the fused polypeptide expressed by one such clone react with a P. falciparum protein that is synthesized late in schizogony and is later present on the surface of the ring-infected erythrocyte. The protein is composed of repeating subunits of 8, 4 and 3 amino acids and is present in all isolates of P. falciparum examined.     

Beta 2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells

Beta 2-microglobulin (beta 2-m) is a highly conserved polypeptide (12,000 molecular weight; 12K) noncovalently associated with the heavy chain (45-48K) of the major histocompatibility complex (MHC) class I antigens. Its synthesis is required for expression of the HLA-A/B and H-2K/D heavy chains at the cell surface; beta 2-m is also associated with the human cell-surface antigens T6 and M241 isolated from thymocytes. However, on the T leukaemic cell line MOLT-4 some of the T6 antigens contain a different 12K subunit, termed beta t (refs 3, 7, 8). Purified human beta 2-m can exchange partially both with human beta 2-m associated with HLA-antigens, and with mouse beta 2-m associated with murine alloantigens. As MOLT-4 cells were grown in the presence of fetal calf serum (FCS) and as serum is known to contain some free beta 2-m, we examined whether beta t was bovine beta 2-m which had replaced endogenous beta 2-m on the surface of the cell. Here we show both that beta 2-m from FCS or human serum (HuS) used in cell culture can exchange with beta 2-m on the cell surface, and that beta t is in fact bovine beta 2-m.     

Bone marrow cells give rise to distinct cell clones within the thymus

The thymus is the major, if not the sole site of maturation of T lymphocytes from their haematopoietic precursors. During embryonic life (at a few well-defined intervals, at least in birds) the thymus receives thymus-homing haematopoietic precursors that give rise to antigen-specific functional T lymphocytes. Although the number and thymic location of distinct T-cell lineages destined to form the peripheral T-cell pool are not yet well defined, at least two independent pathways have been proposed. First, thymic subcapsular lymphoblasts divide and differentiate to give rise to small deep cortical thymic lymphocytes, medullary lymphocytes and thymus emigrants (I.W., unpublished data) and second, the medulla contains an independent self-renewing population that contains the precursors of the peripheral T-cell pool. Following irradiation the thymus may be repopulated by injected haematopoietic cells presumably related to the thymus-homing haematopoietic cells of the embryo. Here we have reconstituted irradiated mice with limiting numbers of bone marrow cells from Thy-1 congeneic donors and have found distinct clones of cells within the thymus. The pattern of reconstitution by the precursor cells indicates that two independent thymus lineages exist: cortex plus medulla, and medulla alone.