Figure 10. Ribosome binding to Sec61p. Wild-type, sec61-3, and srp101-47 cells were grown at 258C and then shifted to 378C for the indicated number of hours. In some cases, cycloheximide was added to the medium when the cultures were shifted to elevated temperature. ER membranes were isolated, washed with high salt, and solubilized with digitonin. The ribosomes were pelleted and the amount of Sec61p in the pellet determined by quantita- tive immunoblotting. Each bar is the average of at least three in- dependent determinations. Some samples were treated with puromycin before solubilization to release nascent polypeptides (column 2). The differences between columns 5 and 6 and col- umns 3 and 4 are statistically significant (P , 0.01). in mammals, the ER network is maintained during mitosis. Yeast cells can therefore serve as an excellent model sys- tem in which to study the mechanisms underlying the for- mation and maintenance of ER networks. Our data indicate that mechanisms proposed for main- taining the reticular structure of the ER in mammalian cells are probably not correct for yeast. We found that mi- crotubules are not required to maintain the structure of the peripheral ER, in contrast to their function in mamma- lian cells. This is perhaps not too surprising, since there are few cytoplasmic microtubules in yeast. While actin does form an extensive cortical network, it is also not required for the maintenance of ER structure. Since there is also relatively little coalignment between actin structures and the ER network, ER tubules are probably stabilized by mechanisms other than association with an underlying cy- toskeleton scaffold. The actin cytoskeleton does appear to be required for ER dynamics, which are drastically re- duced minutes after addition of latrunculin-A to the me- dium. However, it is difficult to rule out that the effect may be indirect. Actin filaments are also required for the dynamics of mitochondria in yeast (Simon et al., 1995), but again the mechanism remains to be elucidated. Given that the nuclear and peripheral ER are structur- ally distinct and that the cytoskeleton does not play a role in maintaining ER structure in yeast, we decided to use ge- netics to search for other components that do. In our vi- sual screen, we looked for mutants that affect either ER inheritance or the maintenance of the ER. Only three out of the thousand mutants we screened had such defects, in- dicating that a general perturbance of cell physiology does not readily lead to these phenotypes. It is clear that the screen has not been saturated, and indeed we have found Figure 11. The SRP receptor mutants also disrupt mitochondrial structure. Cells expressing Sec63-GFP were labeled with TMR- CH2Cl to visualize mitochondria and viewed live. Wild-type cells were grown at 308C. The other strains were grown at 258C and then shifted to 378C for 2 h (srp101-47), 2.5 h (ret1-3), 4 h (mmm1-1), or 30 min (fzo1-1). Arrows point to regions of align- ment of mitochondria and ER tubules in wild-type cells. Images were acquired while focusing on either the center or periphery of the cells. Bars, 5 mm. The Journal of Cell Biology, Volume 150, 2000 472