Figure 8. Phenotypes of vari- ous translocation mutants. A, Images of sec65-1, sec61-3, sec61-2, and sec61-2 Dssh1 cells expressing Sec63-GFP were taken 3 h after shift to nonpermissive temperature (378C). Images were ac- quired while focusing on ei- ther the center or periphery of the cells. Bars, 5 mm. B, Translocation of Kar2p was tested 1 h after shift to the nonpermissive temperature (378C). The cells were pulse- labeled for 7 min and Kar2p was immunoprecipitated. The positions of mature Kar2p (m) and of Kar2p that has not undergone signal sequence cleavage (p) are indicated. Fig. 11 A). In the SRP receptor mutants we isolated, mito- chondria appear to fragment; at nonpermissive tempera- ture, a number of bright spots were seen throughout the cytoplasm (Fig. 11 B). Mitochondrial structure is normal when the mutants are grown at permissive temperature (not shown). Interestingly, when the ER and mitochondria were visualized in the same cell, fragments of the dis- rupted ER were frequently localized next to fragments of mitochondria (Fig. 11 B). Mutants with defects in ER– Golgi complex trafficking that affect ER structure (e.g., ret1-3), also have altered mitochondrial structure. As with the SRP mutants, the disrupted ER and mitochondrial structures are often near one another (Fig. 11 C). Even in wild-type cells, the tubules of the peripheral ER sometimes coaligned with mitochondrial tubules (Fig. 11 A). These data suggest that the membranes of the ER and mitochon- dria are linked at certain sites and that the ER may have an effect on the structure of mitochondria. The reverse may not be true. Conditional mutations in two mitochondrial outer membrane proteins, Mmm1p and Fzo1p, cause mito- chondria to form large spheres (mmm1-1) or fragment (fzo1-1) at nonpermissive temperature (Burgess et al., 1994; Hermann et al., 1998; Rapaport et al., 1998). ER structure is unaffected in these mutants (Fig. 11, D and E). Discussion A major result of this work is the demonstration that the peripheral ER in yeast, previously viewed simply as a bright ring by immunofluorescence (Rose et al., 1989; Preuss et al., 1991), is a tubular network, similar in structure to the ER in higher eukaryotes. The tubules have a relatively constant diameter and undergo the same kinds of dynamic changes that have been described in mammalian cells. As Figure 9. Cycloheximide suppresses both the ER morphology and translocation defects of SRP receptor mutants. Strains expressing Sec63-GFP were grown at 258C and then for 6 h at 378C with or without 1 mM cycloheximide. A, Images of the cells grown with cy- cloheximide. They were acquired while focusing on either the cen- ter or periphery of the cells. Bars, 5 mm. B, A portion of the cultures were pulse-labeled for 7 min and lysates were immunoprecipitated for Kar2p. m, Mature Kar2p; p, precursor of Kar2p. Prinz et al. Mutants Affecting ER Structure 471