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Three proteins required for early steps in the protein secretory pathway also affect nuclear envelope structure and cell cycle progression in fission yeast

Anna Matynia^1,3,*, Sandra S. Salus^2,3,* and Shelley Sazer^1,2,3,

¹ Department of Molecular and Cellular Biology,
² Graduate Program in Cell and Molecular Biology,
³ Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
^* These authors contributed equally to this work

View larger version (81K): [in a new window]   Fig. 1. Amino acid sequences of Sar1p, Sec31p and Pmm1p with their budding yeast and mammalian homologues. (A) Protein sequence alignment of Sar1p in S. pombe (Sp), accession number M95797 (d’Enfert et al., 1992); Sar1p in S. cerevisiae (Sc), accession number A33619 (Nakano and Muramatsu, 1989); and SAR1A in M. musculus (Mm), accession number P36536 (Shen et al., 1993). (B) Protein sequence alignment of Sec31p in S. pombe (Sp), direct submission, accession number CAA17835; Sec31p in S. cerevisiae (Sc), accession number NP_010086 (Goffeau et al., 1996); and the Sec31 protein in H. sapiens (Hs), accession number AAf67836 (Tang et al., 2000). (C) Protein sequence alignment of Pmm1p in S. pombe (Sp), direct submission, accession number AL132984; Sec53p in S. cerevisiae (Sc), accession number NP_011141 (Goffeau et al., 1996); and phosphomannomutase 1 in H. sapiens (Hs), accession number NP_002667 (Hansen et al., 1997). Sequence alignments were generated using ClustalW and displayed using Boxshade. Black boxes represent identities and gray boxes represent conservative substitutions. *The S7N, E72K and G80D mutations in the Sec31-1 protein and the G27S mutation in the Sar1-1 protein are marked with an asterisk.

View larger version (37K): [in a new window]   Fig. 2. sar1-1, sec31-1 and pmm1-1 accumulate ER forms of acid phosphatase. Equal quantities of cell extracts were resolved by SDS-PAGE, transferred to a nylon membrane and probed with the anti-acid phosphatase monoclonal antibody 7B4 (Schweingruber et al., 1986) unless otherwise stated. (A) Wild-type (lanes 1-4) and sar1-1 (lanes 5-8) cells were harvested after 0, 1, 2 or 4 hours at 36°C. (B) Wild-type (lanes 1,2) and sec31-1 (lanes 3,4) cells were harvested after incubation for 0 or 4 hours at 36°C. (C) Wild-type (lanes 1,2) and pmm1-1 (lanes 3,4) cells were treated with cycloheximide for 1 hour at the permissive temperature, washed, and released into pre-warmed media without cycloheximide at 36°C. Untreated cells and cells incubated for 4 hours at 36°C were harvested. (D) Wild-type (lanes 1,2), ypt1-VN (lanes 3,4) and ypt2-VN (lanes 5,6) cells were harvested after 0 and 4 hours at 36°C. Three times as much protein was loaded for the ypt mutants at 25°C to allow better visualization of the acid phosphatase. The arrow indicates the 72 kDa core glycosylated ER form (A,B), the 45-75 kDa incompletely glycosylated ER forms (C) or the high molecular weight Golgi modified forms of acid phosphatase (D).

View larger version (40K): [in a new window]   Fig. 3. sar1 and pmm1 null strains accumulate cytoplasmic membranes. sar1, containing pREP3X-sar1, and pmm1, containing pREP81X-pmm1, were grown in media either without thiamine, to allow expression (promoter ON; A-D) or with thiamine, to repress expression (promoter OFF; E-H). After 48 hours, live cells were stained with both DiOC6, to visualize cellular membranes (A,C,E,G), or Hoechst, to visualize DNA (B,D,F,H). Cells in which the promoter was off contain an increase in cellular membranes compared with cells in which the promoter remained on. Bar, 10 µm.

View larger version (164K): [in a new window]   Fig. 4. sar1-1, sec31-1 and pmm1-1 have accumulated ER membranes and dilated nuclear envelope lumens. Electron micrographs of wild-type (A,E), sar1-1 (B,F), sec31-1 (C,G) and pmm1-1 (D,H) cells grown for 4 hours at 36°C. Wild-type cells contain nuclei with normal morphology and few cytoplasmic membranes. sar1-1, sec31-1 and pmm1-1 have dilated nuclear envelope lumens and accumulated ER membranes (B,C,D, arrows). The nuclear envelopes, indicated by arrowheads, are shown at higher magnification to compare the width of the lumen of wild-type with sar1-1, sec31-1 and pmm1-1 strains (compare E with F,G,H). Bar, 1 µm (A-D); 0.2 µm (E-H).

View larger version (48K): [in a new window]   Fig. 5. Nuclear protein import and export are normal in sar1-1, sec31-1 and pmm1-1. Wild-type, sar1-1, sec31-1 and pmm1-1 cells with integrated GFP-Pap1p under the medium strength nmt1 promoter were incubated at 25°C until mid-log phase, then shifted to 36°C for four hours. Cells were photographed either without addition of hydrogen peroxide, showing that GFP-Pap1p is exported from the nucleus (A,C,E,G) or after exposure to 0.8 mM hydrogen peroxide for 15 minutes, showing that GFP-Pap1p is imported into the nucleus (B,D,F,H). Bar, 10 µm.

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