Figure 5. sec27-95 cells have less peripheral ER at nonpermissive temperature. Thin-section EM of wild- type (A and B) and sec27-95 (C and D) cells grown at 258C (A and C) or 3 h after shift to 378C (B and D). L, Lipid droplet; M, mitochon- drium; N, nucleus; PM, plasma membrane; V, vacu- ole; ER, endoplasmic reticu- lum. Bars, 1 mm. implicated in homotypic ER fusion (Patel et al., 1998). Since homotypic ER fusion may well be required to main- tain ER structure, we looked at a temperature-sensitive mutant in Cdc48p, another protein implicated in the pro- cess (Latterich et al., 1995). Surprisingly, even after four hours at nonpermissive temperature, ER structure re- mained normal in a cdc48-3 mutant (Fig. 4 C). SRP Pathway Disruption Affects ER Structure In the SRP receptor mutants the ER structure was also most disturbed in daughter cells, which often contained one bright fluorescent spot, and in some cases did not con- tain any detectable ER (Fig. 6 A). However, most mother cells also lacked detectable tubular regions of the ER, and instead contained numerous bright fluorescent spots. Ab- normal ER structure was first observed one hour after shift to elevated temperature, and became pronounced af- ter two hours. The structure of the perinuclear ER was not disrupted in the mutants; a ring of Sec63p-GFP was seen around the nucleus stained with 49,6-diamidino-2-phe- nylindole (DAPI; Fig. 6 B). Both SRP receptor mutants have normal ER structure at the permissive temperature Table II. Quantification of the Decrease in Relative ER Surface Area in the Mutants with Disrupted ER Structure Strain FY23 WPY154 WPY152 WPY153 Relevant genotype Wild-type srp101-47 srp102-510 sec27-95 258C 1.36 6 0.12 1.03 6 0.18 1.07 6 0.09 1.01 6 0.10 378C 1.32 6 0.16 0.66 6 0.08 0.26 6 0.04 0.80 6 0.09 The relative ER surface area, mm2 ER (both perinuclear and cortical) per mm3 cell volume, was determined by EM on ultrathin sections of KMnO4-fixed cells grown at 258C, or after a 3-h shift to 378C. Values are the average of 100 cells 6 SD. (Fig. 6 A). We ruled out that the mutants only cause the mislocalization of Sec63-GFP, rather than actually disrupt- ing ER structure, because ss-GFP-HDEL also has an al- tered distribution in the mutants at the nonpermissive temperature (not shown). Analysis of the mutants by thin- section EM revealed a reduction in the amount of ER at the periphery of the cells, particularly at nonpermissive temperature (Fig. 7 and Table II). In addition, large sheets of membrane near the nucleus can be seen that are not present in wild-type cells (Fig. 5). These structures are likely to be responsible for the brightly fluorescing spots seen in the fluorescence microscope. A conditional mutation in SRP itself also affects ER structure. Yeast cells bearing a mutation in Sec65p, the ho- mologue of the mammalian SRP19 subunit (Stirling and Hewitt, 1992), have abnormal ER morphology at the non- permissive temperature, similar to that seen in the srp101- 47 and srp102-510 mutants isolated in the screen (Fig. 8 A). Like other mutants with defects in SRP and SRP recep- tor, srp101-47 and srp102-510 mutants have a protein translocation defect; both mutants accumulate precursor of the ER lumenal protein, Kar2p (Fig. 8 B). However, the translocation defect per se may not be responsible for the altered ER morphology. We tested a number of mutants with temperature-sensitive or cold-sensitive defects in other components of the protein translocation machinery and none of them had abnormal ER structure. Specifi- cally, temperature-sensitive mutations in the major com- ponent of the translocation channel, Sec61p (mutants sec61-3 and sec61-2), have only a few minor changes in ER structure three hours after shift to the nonpermissive tem- perature (Fig. 8 A). These mutants develop a translocation defect less than one hour after shift to elevated tempera- ture (Fig. 8 B; and Rose et al., 1989; Stirling et al., 1992), but continue to grow for a few hours (not shown). We The Journal of Cell Biology, Volume 150, 2000 468