Figure 7. Mutants with con- ditional defects in SRP re- ceptor have altered periph- eral ER morphology at nonpermissive temperature. Thin-section EM of srp102- 510 (A and B) and srp101-47 (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. Bars, 1 mm. shown), whereas those grown in the presence of the drug had almost normal ER (Fig. 9 A). In addition, as described for other translocation mutants (Lee and Beckwith, 1986; Ogg and Walter, 1995), cycloheximide suppressed or re- duced the translocation defect of the mutants (Fig. 9 B). Thus, the role of the SRP pathway in maintaining ER mor- phology is likely related to its known function in targeting ribosomes to the ER membrane. To determine ribosome binding to the ER, we measured the amount of Sec61p that is associated with ribosomes after solubilization of ER membranes in detergent. ER mem- branes from wild-type, sec61-3, and srp101-47 cells were washed with high salt to remove loosely bound ribosomes not engaged in translocation (Gorlich and Rapoport, 1993), and solubilized in digitonin. The ribosomes were pelleted, and the amount of cosedimenting Sec61p was determined by quanti- tative immunoblotting (Fig. 10). At permissive temperature, z30% of the Sec61p was bound to ribosomes in all three strains (Fig. 10, bars 1, 5, and 9). Release of the nascent chains from the ribosomes with puromycin greatly decreased the amount of Sec61p pelleting with the ribosomes (Fig. 10, bar 2; and data not shown), indicating that the association re- quires the presence of a translocating polypeptide. When the strains were grown for three hours at nonpermissive temper- ature, the fraction of Sec61p bound to ribosomes reproduc- ibly decreased to z20% in the srp101-47 strain (Fig. 10, bar 6), whereas it remained about the same in the sec61-3 (Fig. 10, bar 10) and wild-type strains (Fig. 10, bar 3). The amount of Sec61p per cell in these strains was about the same when they were grown at permissive temperature or after three hours at nonpermissive temperature (data not shown; Bied- erer et al., 1996). When the srp101-47 mutant was grown at 378C for prolonged periods, conditions that restore ER mor- phology, the amounts of Sec61p bound to ribosomes in- creased to normal levels (Fig. 10, bar 8 vs. 6). A similar corre- lation between restoration of ER morphology and ribosome binding was seen after treatment of the mutant with cyclo- heximide (Fig. 10, bar 7 vs. 6). A small increase of ribosome- associated Sec61p was observed when wild-type cells were grown with cycloheximide (Fig. 10, bar 4 vs. 3), which might be expected when the rate of translation is slowed. Taken to- gether, these results suggest that the SRP pathway influences ER morphology through its effect on ribosome binding. Connection between ER and Mitochondrial Structures We found that in the SRP receptor mutants, not only pe- ripheral ER, but also mitochondrial structure, is disrupted. In wild-type cells, mitochondria form branched reticular structures near the cell cortex (Hermann and Shaw, 1998; The Journal of Cell Biology, Volume 150, 2000 470