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First published online 7 April 2009
doi: 10.1242/jcs.040949

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A nucleolar protein allows viability in the absence of the essential ER-residing molecular chaperone calnexin

¹ Department of Biochemistry, Université de Montréal, C.P. 6128, succursale Centre-ville, Montréal, Québec H3C 3J7, Canada
² Whitehead Institute for Biomedical Research and Howard Hughes Medical Institute, 9 Cambridge Center, Cambridge, MA 02142, USA

View larger version (27K): [in this window] [in a new window]   Fig. 1. Primary structure of Cif1p. (A) Predicted amino acid sequence of the S. pombe protein encoded by the open reading frame SPCC364.01, as retrieved from GenBank. The predicted NLS is underlined, and basic residues are in bold. (B) Schematic representation of Cif1p. The NLS is in gray, and the quadruple replacement of KRKR27-30 by alanine residues is indicated.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Cincif1 cells display phenotypic differences with Cinhcd_cnx1 cells. (A) Calcofluor staining of Cin cells. Log phase cells (OD595 0.8) were cultured at 30°C or 37°C, and incubated with 20 µg/ml Calcofluor white for 5 minutes. Nomarski-interference images of the same fields of cells stained with Calcofluor are shown above. (B) Sensitivity to cell-wall stress. Exponentially growing cells were adjusted at OD595 0.8, serially diluted (10-1 to 10-4), and spotted on MM plates with or without the indicated chemicals. Plates were incubated at 30°C for 7 days. Results are representative of three independent assays. (C) SDS sensitivity of Cin cells. A volume of 450 µl of a culture at exponential phase (OD595 0.5) was added to 13 ml MM+0.7% agar, and poured on plates containing MM+2% agar. A circle of 3M paper of 5 mm diameter was placed in the center of each plate and 10 µl 10% SDS was added on the circle. Petri dishes were incubated for 7 days at 30°C or at 37°C. The ratio of halos was calculated with respect to WT at 30°C. Results are the average of three independent assays. (D) Metacaspase activity of Cin cells. Metacaspase activity was evaluated by the CaspACE FITC-VAD-fmk fluorescence assay on exponential-phase and 4-day stationary-phase cultures. Stained cells were counted by flow cytometry analysis. Samples of 10,000 cells were analyzed from at least three independent cultures for each strain. Results are the mean of these assays. In (C) and (D), the statistical significance of differences in the results was evaluated by a Student's t-test, pairwise calculated with the lanes indicated. **P<0.01, *P<0.05. Error bars: s.e.m.

View larger version (43K): [in this window] [in a new window]   Fig. 3. cif1+ is unessential for vegetative growth. (A) Tetrad analysis of a cif1+ x cif1::kanMX4 (cif1) diploid. Dissection was performed on MM+AULH, and colonies from germinated spores were streaked on YE+150 µg/ml G418 to verify the presence of the cif1::kanMX4 marker, and on MM+low adenine+ULH to verify the segregation of the ade6 marker. All the tetrads showed 2:2 segregation of both cif1 and ade6 alleles. (B) Northern blot analysis of Cin and cif1 cells. Samples of 5 µg of total RNA prepared from exponential phase cells were loaded on a 1.2% agarose-formaldehyde gel. RNA was probed with a 32P-labeled DNA probe encompassing the entire cif1+ or cnx1+ coding sequence. Photograph of the ethidium-bromide (EtBr) staining shows the 18S and 25S rRNAs as loading control. Cells harboring calnexin on a plasmid have higher levels of cnx1+ RNA; however, these cells contain the same amount of Cnx1p, thus confirming that the levels of plasmid-encoded Cnx1p are equivalent to genomic expression. (C) Western blotting of Cin and cif1 cells. Cell extracts were made as described in the Materials and Methods, and 10 µg of material was loaded for fractionation on a 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane; Western blotting was carried out using rabbit polyclonal anti-Cif1p serum at a 1:5000 dilution, and with anti-Cnx1p polyclonal antibodies at a 1:35,000 dilution. As a loading control, the same membrane was immunoblotted with and anti-tubulin monoclonal antibodies at a 1:5000 dilution.

View larger version (70K): [in this window] [in a new window]   Fig. 4. Nucleolar localization of Cif1p is abolished by the KRKR27-30/AAAA mutation. (A) Cif1p localizes to the nucleolus of WT and both types of Cin cells. Exponentially growing cells expressing a Cif1p-Venus and a Fib1p-mRFP fusion were fixed as described in the Materials and Methods. Slides were mounted with a DAPI-containing media (1 µg/ml DAPI, 1 mg/ml p-phenylenediamine, 90% glycerol). Cells were examined by fluorescence microscopy. Venus, mRFP, DAPI, merge and Nomarski show the same fields of cells for each strain. (B) Replacement of KRKR27-30 with alanines abolishes the nucleolar localization of Cif1p. Exponentially growing cells expressing a Cif1p-KRKR27-30/AAAA-Venus fusion and a Fib1p-mRFP fusion were fixed and analyzed by fluorescence microscopy as in panel A.

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