Table I. Yeast Strains Used in This Study Strain Genotype Source W303 MATa/MATa ade2-1/ade2-1 trp1-1/trp1-1 leu2-3,-112/ leu2-3,-112 his 3-11/his 3-11 ura 3-1/ura 3-1 can1-100/can1-100 Deshaies and Schekman, 1990 FY23 MATa ura3-52 trp1D63 leu2D1 Winston et al., 1995 FY86 MATa ura3-52 his3D200 leu2D1 Winston et al., 1995 RSY455 MATa his4 trp1-1 leu2-3,112 ura3-52 HOL1-1 sec61-3 Stirling et al., 1992 YFP338 MATa leu1-3, 112 ura 3-52 ade2-3 pep4-3 sec61-2 Rose et al., 1989 KF5 MATa leu2-3, 112 ura3-521 ade2-3 pep4-3 sec61-2 ssh1::ADE2 Finke et al., 1996 RSY457 MATa ura3-52 ade2 trp1-1 leu2-3,-112 sec65-1 Stirling et al., 1992 RSY1293 MATa can1-100 leu2-3,-112 his3-11,-15 trp1-1 ura3-1 ade2-1 sec61::HIS3 (pDQ1 [SEC61-his6]) Pilon et al., 1998 RSY1294 RSY1293, but sec61-32 Pilon et al., 1998 RSY1300 RSY1293, but sec61-11 Pilon et al., 1998 RSY1303 RSY1293, but sec61-23 Pilon et al., 1998 RSY1304 RSY1293, but sec61-24 Pilon et al., 1998 SOY162 W303 MATa srp102::HIS3 Ogg et al., 1998 BHY116 MATa trp1 lys2 his3 ura3 ade2srp54::LYS2 Hann and Walter, 1991 SOY36 W303 MATa srp101::ADE2 Ogg et al., 1992 MS177 MATa ura3-52 ade2-101 kar2-159 Vogel et al., 1990 RSY528 MATa leu2 ura3 his4 sec62-1 R. Schekman RSY265 MATa ura3-52 his4-619 sec13-1 R. Schekman RSY267 MATa ura3-52 his4-619 sec16-2 R. Schekman RSY277 MATa ura3-52 sec21-1 R. Schekman RSY281 MATa ura3-52 his4-619 sec23-1 R. Schekman RSY1319 MATa ura3 leu2 his3-D200 trp1 lys2 ade2 sec28::HIS3 R. Schekman RSY769 MATa ura3-52 leu2-3,-112 sec27-1 R. Schekman RSY1317 MATa ura3-52 leu2-3,-112 ret1-3 R. Schekman JSY2977 MATa ura3-52 his3D200 leu2D1 trp1D63 fzo1-1 Hermann et al., 1998 YSB105 MATa trp1D1 leu2 ade2 his3 ura3 lys2 mmm1-1 R. Jensen WPY152 FY23 srp102-510 This study WPY154 FY86 srp101-47 This study WPY153 FY86 sec27-95 This study NY13 MATa ura3-52 P. Novick NY3 MATa ura3-52 sec1-1 P. Novick NY130 MATa ura3-52 sec2-41 P. Novick NY412 MATa ura3-52 sec3-2 P. Novick NY405 MATa ura3-52 sec4-8 P. Novick NY17 MATa ura3-52 sec6-4 P. Novick NY410 MATa ura3-52 sec8-9 P. Novick NY57 MATa ura3-52 sec9-4 P. Novick NY64 MATa ura3-52 sec15-2 P. Novick DBY4060 MATa his4-619 mal gal cdc48-3 D. Botstein WPY107 MATa ura3-52 his4-619 cdc48-3 This study MLY2141 MATa ura3-52 trp1-1 his3-11,-15 ufe::TRP1 (pUT1 [ufe1-1]) M. Latterich 1993). The amount of Sec61p in the fractions was determined by immuno- blotting using an 125I-labeled secondary antibody (NEN Life Science Prod- ucts). All samples were run in triplicate and quantitated using a Fujix Phos- phorImager. Samples were run at various dilutions to ensure that the quantification was performed within the linear range of the PhosphorImager. Results Saccharomyces cerevisiae ER Forms a Tubular Network Previous immunofluorescence microscopy with fixed S. cerevisiae cells only allowed the visualization of the pe- ripheral ER as a bright ring close to the plasma membrane (Rose et al., 1989; Preuss et al., 1991). To obtain a more detailed picture of the ER, we used a fusion of the ER pro- tein Sec63p to GFP (Sec63-GFP) and employed high-reso- lution fluorescence microscopy with live cells. A stack of images with focal planes 0.2-mm apart were obtained with a wide-field optical sectioning microscope and decon- volved. Two of the images from each stack are shown in Fig. 1, with focus planes close to the center and periphery of the cell. With the improved resolution, we were able to demonstrate that the peripheral ER forms a network of in- terconnected tubules (Fig. 1, bottom left). This network could be seen on either side of the cell in only one or two image planes close to the plasma membrane. The nuclear ER, on the other hand, forms a continuous membrane (Fig. 1, top left). To confirm that the peripheral network is ER, we used a GFP-fusion protein targeted to the ER lu- men. We expressed GFP with an NH2-terminal signal sequence and a COOH-terminal ER retention signal (HDEL; Pelham et al., 1988). This fusion protein, called ss-GFP-HDEL, shows a localization pattern similar to Sec63-GFP (Fig. 1, right). Thus, the peripheral ER in yeast looks strikingly similar to the ER network in mammalian cells. The network was seen at all stages of the cell cycle and appeared in the daughter cell as soon as the bud started to form. The ER network is also thought to be maintained during mitosis in higher eukaryotes (Ellenberg et al., 1997). Prinz et al. Mutants Affecting ER Structure 463