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      Identification of Yeast Genes Involved in K + Homeostasis: Loss of Membrane Traffic Genes Affects K + Uptake

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          Abstract

          Using the homozygous diploid Saccharomyces deletion collection, we searched for strains with defects in K + homeostasis. We identified 156 (of 4653 total) strains unable to grow in the presence of hygromycin B, a phenotype previously shown to be indicative of ion defects. The most abundant group was that with deletions of genes known to encode membrane traffic regulators. Nearly 80% of these membrane traffic defective strains showed defects in uptake of the K + homolog, 86Rb +. Since Trk1, a plasma membrane protein localized to lipid microdomains, is the major K + influx transporter, we examined the subcellular localization and Triton-X 100 insolubility of Trk1 in 29 of the traffic mutants. However, few of these showed defects in the steady state levels of Trk1, the localization of Trk1 to the plasma membrane, or the localization of Trk1 to lipid microdomains, and most defects were mild compared to wild-type. Three inositol kinase mutants were also identified, and in contrast, loss of these genes negatively affected Trk1 protein levels. In summary, this work reveals a nexus between K + homeostasis and membrane traffic, which does not involve traffic of the major influx transporter, Trk1.

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          Transformation of intact yeast cells treated with alkali cations.

          Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication.
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            Receptor downregulation and multivesicular-body sorting.

            The sorting of proteins into the inner vesicles of multivesicular bodies is required for many key cellular processes, which range from the downregulation of activated signalling receptors to the proper stimulation of the immune response. Recent advances in our understanding of the multivesicular-body sorting pathway have resulted from the identification of ubiquitin as a signal for the efficient sorting of proteins into this transport route, and from the discovery of components of the sorting and regulatory machinery that directs this complex process.
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              The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls.

              Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-beta-cyclodextrin (MbetaCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MbetaCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.
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                Author and article information

                Contributors
                Role: Communicating editor
                Journal
                G3 (Bethesda)
                ggg
                ggg
                ggg
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                2160-1836
                1 June 2011
                June 2011
                : 1
                : 1
                : 43-56
                Affiliations
                [1]Department of Biology, Georgetown University, Washington, DC 20057
                Author notes
                [1]

                Present address: Harvard Medical School, Boston, MA 02115.

                [2]

                Present address: Shenandoah University Bernard J. Dunn School of Pharmacy, Ashburn, VA 20147.

                [3]

                Present address: Emory University Rollins School of Public Health, Atlanta, GA 30322.

                [4 ]Corresponding author: Department of Biology, Georgetown University, 406 Reiss Science Center, Box 571229, Washington, DC 20057-1229. E-mail rosenwaa@ 123456georgetown.edu
                Article
                GGG_000166
                10.1534/g3.111.000166
                3276120
                22384317
                376b0c47-3f2c-4031-8c10-cf6811b7e32d
                Copyright © 2011 Fell et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution Unported License ( http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 9 December 2010
                : 24 March 2011
                Categories
                Investigation
                Custom metadata
                v1

                Genetics
                vps genes,trk1
                Genetics
                vps genes, trk1

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