Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.     

The detection of 5-lipoxygenase and cyclo-oxygenase products in sputum of patients with chronic bronchitis and bronchiectasis

Leukotrienes (LTs) and prostanoids (Ps) were detected in sputum of patients with chronic bronchitis and/or bronchiectasis (CB/B) using selective superfusion bioassay and radioimmunoassay (RIA) techniques. Analysis of sputum extracts showed a 4-fold increase in the level of LTB4 compared to the cysteinyl-containing LTs (LTC4/LTD4). The measurement of cyclo-oxygenase products (COPs) indicated relatively greater amounts of the vasodilator prostaglandin E2 (PGE2) and prostacyclin (PGI2) compared to the vasoconstrictor prostaglandin F2 alpha (PGF2 alpha) and thromboxane A2 (TxA2) agents (70:30% of total COPs respectively). The presence of eicosanoids (LTs and Ps) in sputum of patients with CB/B suggest that these biologically active substances may act as mediators of bronchoconstriction and inflammation in these diseases.     

Appearance of alpha-smooth muscle actin in human eye lens cells of anterior capsular cataract and in cultured bovine lens-forming cells

Using light and electron-microscopic immunolocalization techniques, and gel electrophoresis combined with immunoblotting, we have examined the expression of cytoskeletal proteins in normal human fetal, child and adult lenses, in human anterior capsular cataract and in bovine lens cells in vivo and in vitro. In this report, we focus our observations on the pattern of actin-isoform expression during normal and pathological situations in vivo and culture conditions. We have noted that cells of developing and mature human lenses as well as bovine lens cells in situ contain only beta- and gamma-actins. In contrast, alpha-smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle differentiation, was demonstrated in bovine lens cells at different times of culture. Moreover, the multilayered cells observed in the subcapsular zone of human anterior capsular cataract were characterized by the presence of alpha-sm actin. Thus, extensive changes in actin-isoform expression take place in lens cells growing in culture and may also occur during cataractogenesis. The biological meaning of the appearance of a marker of myoid differentiation in the ectodermally derived lens-forming cells is discussed.     

Impaired natural killer cell function in hemophiliacs with or without continuous substitution

In the present study, 28 hemophiliacs substituted continuously and 5 hemophiliacs who had received almost no blood products were investigated. Cells of OKT 3+, OKT 4+, and OKT 8+ subsets were counted. Percoll separated fractions of peripheral blood mononuclear cells were examined by morphological criteria and were tested for NK cell activity. We found that the NK cell activity of both groups of hemophiliacs was decreased on testing Ficoll separated cells or low density Percoll separated cells. Normal NK cell activity was found in medium density cells of hemophiliacs. Two possible explanations are discussed: first, the NK cell activity may be suppressed in hemophiliacs and secondly, there may be a block in maturation of NK cell activity. It is unlikely that chronic substitution by blood products counts for these alterations. The possible role of chronic infections is discussed.     

Relation between enzyme release and metabolic changes in reversible anoxic injury of myocardial cells

Cultured adult cardiac myocytes were exposed to anoxia under substrate-free conditions. When compared to the metabolic changes in the oxygen deficient organ, those in the anoxic cell culture proceed in a similar, yet prolonged manner. Release of cytosolic enzymes starts with minor energetic disturbances and proceeds in close correlation to the actual ATP decay. Below 2 μmol. ATP/g_WW, an increasing number of cells becomes irreversibly damaged, but above, 30 min reoxygenation leads to extensive recovery of the whole preparation. The results indicate that leakage of cytosolic enzymes during the early stage of anoxia is due to a gradual protein release from the individual cells, related to reversible membrane alterations.     

THE DIFFERENTIAL SENSITIVITY OF CULTURED CHICK MESODERMAL CELLS TO ACTINOMYCIN D

Cells were isolated from the somite mesoderm and from the unsegmented (presomite) mesoderm of early chick embryos and exposed to actinomycin D in single cell culture. Actinomycin D inhibited proliferation in cell cultures derived from the unsegmented mesoderm, although the same concentrations of this antibiotic did not inhibit cultures derived from the somite mesoderm. This differential sensitivity parallels the regionally specific necrosis and degeneration observed in the unsegmented mesoderm of intact chick embryos exposed to actinomycin D. In culture, both cell types exhibited approximately the same permeability to labeled actinomycin D and showed comparable inhibition of RNA, DNA, and protein syntheses in the presence of the antibiotic. However, freshly isolated mesodermal cells from the somite region had a higher content of RNA than did cells from the unsegmented region, and the somite cells maintained a higher rate of macromolecular synthesis in untreated cultures.     

Biological control of the wetlands weed purple loosestrife (Lythrum salicaria) in the Pacific northwestern United States

Purple loosestrife (Lythrum salicaria) is an Eurasian perennial hydrophyte that has become naturalized in wetlands and in and along waterways throughout temperate North America. The ecological integrity of such areas is threatened by rapidly forming monotypic infestations that displace valued flora and diminish critical fish and wildlife habitat. The inability of physical, cultural, and chemical methods to provide adequate control of the weed has led to the development of an insect-based biological control program. The first field releases of the bud and leaf feeding beetles, Galerucella calmariensis and G. pusilla, and a root-mining weevil, Hylobius transversovittatus, were made in the United States and Canada in 1992. A total of 4740 Galerucella spp. adults were released in central Washington during 1992 and 1993 at eight sites and 471 H. transversovittatus egg inoculations were made in 1993 at three locations. Establishment of both Galerucella spp. was confirmed and Hylobius colonization was achieved.     

The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.