[PMC free article] [PubMed] [Google Scholar]Jenne N., Frey K., Brugger B., Wieland F. proteins to the cytosol and partial resistance of the BL21 and purified by glutathione-Sepharose 4B column chromatography according to the manufacturer’s teaching (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). The antiserum was affinity-purified by sequential adsorption to Affigel 15 (Bio-Rad, Hercules, CA)-immobilized GST and GST-Surf4 followed by acid elution. Cell Tradition HeLa cells and HeLa cells stably expressing green fluorescent protein (GFP)-ERGIC-53 (Ben-Tekaya for 1 h, followed by solubilization in 25 mM bis-Tris-HCl, pH 7.0, 2% digitonin, and 500 mM 6-amino-caproic acid. The lysates were cleared at 100,000 for 1 h, and then they were separated by Blue Native-PAGE (Hunte for 1 h. Supernatants were incubated with anti-ERGIC-53 Benzenesulfonamide and anti-HA antibodies covalently coupled via dimethyl pimelimidate to protein A-Sepharose beads (Harlow and Lane, 1999 ) or with anti-p23 and anti-p24 antibodies bound to protein A-Sepharose. Beads were washed four instances in 50 mM Tris-HCl, pH 7.4, 0.1% digitonin, 150 mM NaCl, and 2 mM CaCl2. Proteins were separated by SDS-PAGE, and then they were transferred to nitrocellulose membranes for immunoblotting. Small Interfering RNA Transfection siRNA oligos were purchased from Eurogentec (Seraing, Belgium) and QIAGEN (Venlo, The Netherlands). Three siRNA oligonucleotides (oligos) were designed against Surf4 and two against p25. siRNA oligos for ERGIC-53 knockdown were explained previously (Nyfeler test. Immunoblotting Cells were lysed for 1 h at 4C in PBS comprising 1% digitonin, supplemented Benzenesulfonamide with protease inhibitors. Lysates were centrifuged at 20,000 for 30 min at 4C. Forty micrograms of protein per lane were separated by SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted sequentially with main and secondary antibodies, and visualized by enhanced chemiluminescence (GE Healthcare). Metabolic Labeling HeLa cells were deprived of l-methionine for 20 min, pulsed for 10 min with 100 Ci of [35S]methionine (PerkinElmer Existence and Analytical Sciences, Boston, MA), and chased for the indicated instances in HeLa tradition medium comprising 10 mM l-methionine. At the end of the chase, the medium was collected and centrifuged for 10 min at 10,000 to remove cell debris. For the 0-min chase time, cells of parallel cultures were homogenized in PBS by passing them 10 instances through a 25-gauge needle. Five-microliter aliquots of homogenate and press were trichloroacetic acid (TCA)-precipitated, and radioactivity was measured by scintillation counting. Total protein secretion into the medium was normalized to the total counts in cell homogenates at 0-min chase. Osmotic Stress Treatment HeLa cells cultivated on 18-mm glass coverslips were incubated in 37C hypotonic medium (60 mM NaCl, 20 mM Benzenesulfonamide HEPES, pH 7.4, and 2.5 mM MgOAc) for 5 min at 37C. The cells were washed Rabbit polyclonal to ATF5 two times in ice-cold PBS, and then they were fixed on snow using 3% paraformaldehyde and processed for immunofluorescence microscopy. RESULTS Human Surf4 Localizes to the ERGIC and Cycles in the Early Secretory Pathway Although found out quite some time ago, mammalian Surf4 remains mainly uncharacterized. Actually its subcellular localization is definitely uncertain. N-terminally tagged Surf4 localizes to the ER, whereas C-terminally tagged Surf4 localizes to the Golgi in transfected cells (Reeves and Benzenesulfonamide Fried, 1995 ). In contrast, endogenous Surf4 was recognized by mass spectrometry in an ERGIC portion isolated BFA-treated HepG2 cells (Breuza topology (Number 3C). Therefore, the changes in Golgi morphology induced by a knockdown of Surf4 and ERGIC-53 or p25 are indistinguishable by both light and electron microscopy. Cargo Receptor Silencing Destabilizes the ERGIC without Influencing ER Exit Sites or Protein Secretion The finding that Benzenesulfonamide a double knockdown of Surf4 and ERGIC-53 and a single knockdown of p25 induced a Golgi phenotype was unpredicted because all three proteins are primarily associated with the ERGIC, although they also cycle through the Golgi to some extent (Schweizer test, p 0.05). Results are means SD (n = 8). Collectively, the morphological, biochemical, and live cell imaging results indicate that cargo receptor silencing destabilizes the ERGIC without initial impairment of overall protein secretion..