vitro formation of ER-like tubule networks in the absence of microtubules has recently been reported (Dreier and Rapoport, 2000). In addition, maintenance of ER struc- ture does not solely rely on a microtubule network, since ER tubules frequently do not coalign with it (Terasaki et al., 1986; Lee et al., 1989). In plant cells, actin filaments, rather than microtubules, are thought to be important for ER formation and maintenance (Terasaki, 1990; Liebe and Menzel, 1995). Little is known about the structure of ER in Saccharo- myces cerevisiae. Indirect immunofluorescence and EM have shown that the yeast ER consists primarily of perinu- clear and cortical domains, with a few tubules connecting them (Matile et al., 1969; van Rijn et al., 1975; Novick et al., 1980; Rose et al., 1989; Preuss et al., 1991). Structural de- tails of the cortical ER, which is closely apposed to the plasma membrane, remain to be elucidated. In addition, it is unclear which mechanisms or molecules are involved in keeping the ER at the cell periphery, and whether the structure of the peripheral ER remains intact during the cell cycle. Here, we show that the peripheral ER forms a dynamic tubular network throughout the cell cycle and have isolated mutants that dramatically affect ER struc- ture. Materials and Methods Media, Plasmids, and Strains The strains used are listed in Table I. Media were prepared according to standard methods (Sherman, 1991). The plasmid pJK59 encodes Sec63- GFP, a fusion of the S65T V163A mutant of GFP to the COOH terminus of Sec63p under the SEC63 promoter. It was constructed by amplifying SEC63 and z400 bp of its 59 promoter from pPS599 (from R. Schekman, University of California, Berkeley, CA) with primers 59-GGTCTA- GACTTCAAGTTCCCAGCGAG-39 and 59-GGCTCGAGCTTCTGG- TGATTCATCATCTTC-39. The resulting product was cloned between the XbaI and XhoI sites of the CEN/URA3 plasmid, pJK50 (Kahana et al., 1998). The plasmid pWP1055 encodes ss-GFP-HDEL under the MET25 promoter. The DNA encoding green fluorescent protein (GFP)1 was am- plified from pJK59 using oligonucleotides that added sequences encoding the signal sequence of carboxypeptidase Y at the NH2 terminus (59- AGATACTCTAGAATGAAAGCATTCACCAGTTTACTATGTGG- ACTAGGCCTGTCCACTACACTCGCTAAGCCGCTAGCAAAGG- AGAAGAACTC-39) and HDEL at the COOH terminus of GFP (59- GATATACGAATCCCAATTCGTCGTGGCAGCCGGATCCTTTG- TATAGTTC-39). The product was then cloned into the CEN/URA3 plas- mid p416MET25 (Mumberg et al., 1994) between the XbaI and EcoRI sites. Isolation and Characterization of Mutants with Disrupted ER Structure A collection of 1,016 mutants with growth defects at 378C was obtained from C. Cole (Dartmouth Medical School, Hanover, NH; Amberg et al., 1992) and transformed with pJK59. This plasmid could not be introduced into 24 of the mutants. The mutants containing pJK59 were grown at 258C, were shifted to 378C for 3 h, and were then examined live in a ZEISS Ax- ioplan II fluorescence microscope using a ZEISS 1003 Plan Apochromat oil immersion objective. No fluorescent signal could be detected in 27 of the mutants after the shift to nonpermissive temperature. Microscopy Cells expressing GFP were mounted in growth medium and examined live. For time course experiments, coverslips were sealed with Valap. Im- 1Abbreviations used in this paper: GFP, green fluorescent protein; SRP, signal recognition particle. ages were acquired using a DeltaVision microscope (Applied Precision Instruments) built around a ZEISS Axiovert microscope with a PXL CCD camera (Photometrics) and a ZEISS 1003 Plan Apochromat oil immer- sion objective. All images shown were deconvolved and scaled using Delta Vision software. To stain the actin cytoskeleton with Alexa 594 phalloidin (Molecular Probes), cells were fixed in 4% formaldehyde, 50 mM KPO4, pH 6.5, and 1 mM MgCl2 for 2 h at room temperature. They were then washed once with PBS pH 7.4 and resuspended in PBS at 10 OD600 /mL. 92.4 mL of the cell suspension was mixed with 7.6 mL of Alexa 594 phalloidin (6.6 mM stock in methanol) and incubated for 1 h at room temperature in the dark. The cells were washed three times with cold (48C) PBS and viewed imme- diately. Immunofluorescence with antitubulin antibody and rhodamine-conju- gated secondary antibody (Molecular Probes) was performed as described in Pringle et al. (1991). For Fig. 6 B, the cells were grown for 1 h in medium supplemented with 0.1 mg/ml DAPI to visualize nuclear DNA. Mitochondria were stained with TMR-CH2-Cl (Mitotracker, Molecular Probes) as described in Nunnari et al. (1997). Electron Microscopy Cells were fixed in 1.5% KMnO4 for 20 min, poststained with uranylace- tate for 4 h, and were dehydrated by incubation with 50, 70, 96, and 100% ethanol for 15 min each. The cells were then incubated in fresh 100% eth- anol for 30 min and embedded in Epon 812. Ultrathin sections were cut with a diamond knife and examined in a Philips EM 400. The relative sur- face area of the ER was calculated with the point counting technique (Weibel and Bolender, 1976). The final magnification of the micrographs was 6,000 and the point spacing of the test system was 1.5 cm. Pulse-labeling and Immunoprecipitation of Kar2p Pulse-labeling and immunoprecipitation were done essentially as de- scribed in Stirling et al. (1992), except that 5 OD600 units of cells were la- beled for 7 min with 250 mCi of 35S protein-labeling mix (NEN Life Sci- ence Products). Treatment of Cells with Cycloheximide, Nocodazole, and Latrunculin-A For the experiments with cycloheximide, cultures were grown logarithmi- cally at 258C, 1 mM cycloheximide (Sigma-Aldrich) was added to half of each culture, and the cultures were then grown at 378C for 6 h. This amount of cycloheximide decreased the growth rate of wild-type cells about fivefold and did not significantly affect the growth rate of the srp101-47 and srp102-510 mutants. A portion of the cultures was pulse- labeled and Kar2p was immunoprecipitated as described above. To depo- lymerize microtubules, cells were grown with 20 mg/mL nocodazole (1 mg/ mL stock in DMSO; Sigma-Aldrich) for 2.5 h (Jacobs et al., 1988). Latrun- culin-A (Molecular Probes) was added to cultures at 200 mM from a 10 mM stock in DMSO (Ayscough et al., 1997), and cultures were grown for the indicated amount of time. Ribosome Binding to Sec61p The fraction of Sec61p bound to ribosomes was determined basically as described in Panzner et al. (1995). Cultures were grown in YPD to an OD600 of 0.5 at 258C, and then half of each culture was shifted to 378C for 3 h while the rest continued to grow at 258C. NaN3 was then added to 10 mM and the cultures cooled in an ice-water bath. The cells were pelleted and resuspended with an equal volume of buffer containing 50 mM potassium acetate, 10 mM magnesium acetate, 20% glycerol, and 100 mM Hepes, pH 7.8, and lysed in a Biospec bead beater. The lysates were spun at 4,000 g to remove unbroken cells and debris, and then at 12,000 g to remove mito- chondria and nuclei. Microsomes were pelleted by spinning at 500,000 g for 30 min. The membranes were washed in buffer containing 800 mM po- tassium acetate, 10 mM magnesium acetate, 10% glycerol, and 50 mM Hepes, pH 7.8, and resuspended in this buffer. Microsomes were solubi- lized by adding digitonin to 2.5% and incubating at 48C for 30 min. Ribo- somes were pelleted by spinning at 500,000 g for 30 min. To release na- scent chains from the microsomes, 2 mM puromycin (Fluka) and 1 mM GTP were added before solubilization with digitonin and the microsomes were incubated for 1 h on ice and 30 min at 308C (Gorlich and Rapoport, The Journal of Cell Biology, Volume 150, 2000 462