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The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

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      Abstract

      We have identified CALNUC, an EF-hand, Ca 2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gα i3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gα i3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca 2+-binding protein detected by 45Ca 2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.

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      Most cited references 54

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      High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.

      A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.
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        Characterization of a cis-Golgi matrix protein, GM130

        Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER- Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.
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          Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum

          A rapid and simple method for the isolation of membranes from subcellular organelles is described. The procedure consists of diluting the organelles in ice-cold 100 mM Na2CO3 followed by centrifugation to pellet the membranes. Closed vesicles are converted to open membrane sheets, and content proteins and peripheral membrane proteins are released in soluble form. Here we document the method by applying it to various subfractions of a rat liver microsomal fraction, prepared by continuous density gradient centrifugation according to Beaufay et al. (1974, J. Cell Biol. 61:213-231). The results confirm and extend those of previous investigators on the distribution of enzymes and proteins among the membranes of the smooth and rough endoplasmic reticulum. In the accompanying paper (1982, J. Cell Biol. 93:103-110) the procedure is applied to peroxisomes and mitochondria.
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            Author and article information

            Affiliations
            Division of Cellular and Molecular Medicine and Department of Pathology, University of California, San Diego, La Jolla, California 92093-0651
            Author notes

            Address correspondence to Marilyn G. Farquhar, Ph.D., Division of Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0651. Tel.: (619) 534-7711. FAX: (619) 534-8549. E-mail: mfarquhar@ 123456ucsd.edu

            Journal
            J Cell Biol
            The Journal of Cell Biology
            The Rockefeller University Press
            0021-9525
            1540-8140
            29 June 1998
            : 141
            : 7
            : 1515-1527
            2132997
            9647645
            Categories
            Articles

            Cell biology

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