Figure 6. Mutants with al- tered ER morphology at the nonpermissive temperature. A, Images of srp101-47 and srp102-510 cells expressing Sec63-GFP grown at either 258C or 2 h after shift to nonpermissive temperature (378C). Arrows point to the daughter cells in some im- ages. Images were acquired while focusing on either the center or periphery of the cells. Bars, 5 mm. B, srp101- 47 cells grown for 2 h at 378C and stained with DAPI to vi- sualize nuclear DNA (blue). Bar, 5 mm. ruled out that differences in strain background account for the difference between the sec61 mutants and the SRP pathway mutants, by moving the sec61 alleles into the same strain background as the SRP receptor mutants we isolated (data not shown). ER structure is also not signifi- cantly disrupted in cold-sensitive sec61-11, sec61-23, sec61- 24, or sec61-32 mutants after they were grown at nonper- missive temperature for up to six hours (not shown). A sec61-2 mutant that also lacks the Sec61p homologue Ssh1p (Dssh1 sec61-2) has a strong protein translocation defect (Finke et al., 1996), but no ER morphology defect (Fig. 8 A). We also looked at two mutants with tempera- ture-sensitive defects in proteins required for posttransla- tional translocation of proteins into the ER, a process that does not require the SRP pathway (Panzner et al., 1995). Both mutants (kar2-159 and sec62-1) had only slight ER morphology alterations similar to those seen in the sec61 mutants (not shown). Thus, it seems that the altered ER morphology is not a consequence of a general transloca- tion defect. Neither SRP101 nor SRP102 are essential genes, but cells lacking them grow approximately five times slower than wild-type cells (Ogg et al., 1992, 1998). The srp101-47 and srp102-510 mutants we isolated were also able to grow slowly at the nonpermissive temperature. After prolonged growth at 378C (.24 h) the ER morphology defect was no longer observed (not shown). Similarly, cells with dele- tions of SRP101, SRP102, or SRP54 (which encodes a component of SRP), showed no ER morphology defect (not shown). One way the SRP pathway might affect ER structure is via ribosome targeting. We reasoned that slowing transla- tion might restore ribosome binding in mutants with a de- fect in the SRP pathway by giving ribosome–nascent chain complexes more time to bind to the ER membrane. Suble- thal doses of the translation elongation inhibitor cyclohex- imide were added to cultures of wild-type, srp101-47, and srp102-510 strains immediately before shifting them to the elevated temperature. After six hours, the mutants grown without cycloheximide had ER morphology defects (not Prinz et al. Mutants Affecting ER Structure 469