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doi: 10.1242/10.1242/jcs.00187

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Sbh1p, a subunit of the Sec61 translocon, interacts with the chaperone calnexin in the yeast Yarrowia lipolytica

Laboratoire de Génétique moléculaire et cellulaire, INRA, CNRS, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France

View larger version (26K): [in a new window]   Fig. 4. Intracellular level of precursor forms of the alkaline extracellular protease in Y. lipolytica wild-type and sbh1 strains. Total intracellular protein was extracted from wild-type cells (lane 1), wild-type cells transformed with the mutated copy of the XPR2 gene (lanes 2 and 3), sbh1 cells (lane 4) and sbh1 cells expressing the mutant protease (lanes 5 and 6). Proteins were analyzed by SDS-PAGE and blotted with anti-AEP antibodies.

View larger version (18K): [in a new window]   Fig. 1. Alignment of the Y. lipolytica Sbh1p protein and the two Sbh2p and Sbh1p proteins from S. cerevisiae. Identical amino acids in the three proteins are in black. Identical amino acids in YlSbh1p and ScSbh2p or YlSbh1p and ScSbh1p are in red and blue.

View larger version (73K): [in a new window]   Fig. 2. (A) Comparison at 28°C on rich medium of the growth and colonies morphology of the Y. lipolytica wild-type and sbh1 strains. (B) Comparison of the SDS resistance of the Y. lipolytica wild-type and sbh1 strains.

View larger version (20K): [in a new window]   Fig. 3. Levels of Kar2p in Y. lipolytica wild-type and sbh1 strains treated with tunicamycine. Exponential cultures were treated for 3 hours with 10 µg/ml tunicamycine before intracellular protein extraction. Same amounts of total protein were separated by SDS-PAGE, transferred onto nitrocellulose and blotted using anti-kar2p antibodies.

View larger version (14K): [in a new window]   Fig. 5. (A) Schematic representation of the different genomic fragments encoding the Y. lipolytica calnexin protein selected in the two-hybrid library screen using the Gal4BD-Sbh1p fusion protein as the bait. The position of the fusion point, relative to the initiator codon of YlCNX1, with the Gal4p activating domain is indicated. (B) Schematic representation of the different deletions of Cnx1p tested for their interaction with Sbh1p in the two-hybrid assay.

View larger version (62K): [in a new window]   Fig. 6. (A) Nucleotidic sequence of the CNX1 gene from Y. lipolytica and amino acid translation. The start and stop codons are in bold and the SacI (positions 1207 and 1743), SalI (position 1396) and XhoI sites (positions 406 and 1432) are both in italic and underlined. The signal sequence (amino acids 1 to 18) and the transmembrane helix (amino acids 495 to 524) are in bold. The different fusion points found in the two-hybrid library are both in bold and underlined.

View larger version (91K): [in a new window]   Fig. 6. (B) Amino acid alignment of YlCnx1p and calnexins from Aspergillus nidulans (ANCNX), Schizosaccharomyces pombe (SPCNX), Homo sapiens (HSCNX) and Saccharomyces cerevisiae (SCCNX).

View larger version (60K): [in a new window]   Fig. 7. Analysis of the interacting domain of Sbh1p with Cnx1p using the two-hybrid system. All diploids are able to grow on medium A, and only interaction between Sbh1p and Cnx1p allows growth on medium B (expression of two reporter genes is required). 1 and 2, pAS2SBH1 + pGADCNX1 (entire protein); 3, pAS2SBH1SalI (deletion of the C-terminal domain) + pGADCNX1; 4, pAS2 + pGADCNX1.

View larger version (17K): [in a new window]   Fig. 8. (A) Co-precipitation of Cnx1p with Sbh1p. A membrane-enriched fraction of the strain expressing the HA-tagged Sbh1p or wildtype was prepared by centrifugation of a total protein extract for 30 minutes at 18,000 g. For solubilization, the pellet was resuspended in phosphate buffer saline and 2% Triton X-100 was added. The sample was further clarified by a centrifugation at 18,000 g. 5 µl of the solubilized supernatant was directly resolved by SDS-PAGE (lane 1) and compared to an anti-HA immunoprecipitate (corresponding to a 100 µl aliquot of the solubilized fraction) from the epitope-tagged strain (lane 2) and from the wild-type strain (lane 3). A western blot analysis was then done using anti-Cnx1p antibodies. (B) Precipitation of Cnx1p with Sec61p or Sec62p. A membrane-enriched fraction of the strain expressing the c-myc-tagged Sec61p was prepared by centrifugation of a total protein extract for 30 minutes at 18,000 g. For solubilization, the pellet was resuspended in phosphate buffer saline and 2% Triton X-100 was added. The sample was further clarified by a centrifugation at 18,000 g. 5 µl of the solubilized supernatant was directly resolved by SDS-PAGE (lane 1) and compared to an anti-c-myc (lane 2) or an anti-Sec62p immunoprecipitate (lane 3) corresponding to a 100 µl aliquot of the solubilized fraction. A western blot analysis was then done using anti-Cnx1p antibodies. (C) Co-precipitation of YlCnx1p with ScSbh2p. A membrane-enriched extract of the strain expressing the ScSbh2p was solubilized in 2% Triton X-100 and centrifuged at 18,000 g for 30 minutes. Proteins in the supernatant were either directly analyzed by SDS-PAGE (lane 1) or first immunoprecipitated in the presence of anti-ScSbh2p antibodies (lane 2) before western blotting using anti-Cnx1p antibodies.

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