Figure 3. Neither actin fila- ments nor microtubules are required to maintain cortical ER structure. A, Images of the ER in wild-type cells ex- pressing Sec63-GFP were taken after 2.5 h of growth in medium with either 20 mg/ml nocodazole or 200 mM la- trunculin-A. Images were ac- quired while focusing on ei- ther the center or periphery of the cells. Bars, 5 mm. B, Wild-type cells expressing Sec63-GFP were fixed and stained with Alexa 594 phal- loidin to visualize actin. Im- ages were acquired while fo- cusing on the cell cortex. Arrows point to regions in which actin filaments and ER tubules align. Bars, 5 mm. sequenced srp101-47 and srp102-510. The alteration in Srp101p (L422P) affects a residue that is not conserved be- tween species, but is close to two regions likely to be in- volved in GTP hydrolysis (Ogg et al., 1992; Montoya et al., 1997). The change in Srp102p (L58P) is in a region of un- known function. ER–Golgi Trafficking Mutants Affect ER Structure In the sec27-95 mutant found in the screen, the peripheral ER in mother cells was disorganized and appeared to be converted from tubules to sheets. In z50% of the cells, the ER in daughter cells was dramatically reduced and disor- ganized (Fig. 4 A). The phenotype was not simply the re- sult of mislocalization of Sec63-GFP, since ss-GFP-HDEL showed a similar distribution (not shown). In thin-section EM, there was a reduction in the amount of peripheral ER at nonpermissive temperature in both mother and daugh- ter cells. Only small areas of the peripheral ER were seen, in contrast to the situation in wild-type cells in which most of the plasma membrane has ER tubules near it (Fig. 5). To quantitate these differences, we determined the surface area of the ER (both perinuclear and cortical) relative to total cell volume. There was a significant decrease in the relative surface area in sec27-95 cells compared with wild- type cells which became more pronounced at nonpermis- sive temperature (Table II). A similar phenotype was shared by mutants with condi- The Journal of Cell Biology, Volume 150, 2000 466