Figure 2. Actin is required for the dynamics of the pe- ripheral ER. Time-lapse im- ages of cortical ER in wild- type cells expressing Sec63- GFP. A, Images of a cell taken at 3-min intervals (mother cell on bottom). Bar, 5 mm. To facilitate com- parisons between frames, stars have been placed in identical places in each frame. B, Images of a portion of a cell taken at 5-s inter- vals. Indicated are an in- stance of ring closure (a) and formation of a new ER tu- bule (b). Bar, 1 mm. C, Im- ages of a cell taken at 3-min intervals 10 min after 200 mM latrunculin-A was added to the medium (mother cell on bottom). Bar, 5 mm. same portion of each image of the daughter cell, and calcu- lated an average correlation coefficient. The coefficient for the drug-treated cell (0.779) was clearly higher than for the control cell (0.293), and may have been underesti- mated because of a slight drift of the sample or Brownian movement. Taken together, our results suggest that the cytoskeleton is not required for maintenance of the ER structure, but actin filaments play a role in ER dynamics. Isolation of Mutants with Disrupted ER Structure We used a genetic screen to identify proteins that are re- quired for maintenance of the structure of the cortical ER. Since maintaining ER structure may be essential for cell viability, we screened a collection of temperature-sensitive mutants (Amberg et al., 1992) for those that had altered cortical ER structure or defects in segregating the periph- eral ER into the daughter cell. Approximately 1,000 mu- tants were transformed with the plasmid encoding Sec63- GFP. They were grown at permissive temperature (258C), shifted to nonpermissive temperature (378C) for three hours, and examined in a fluorescence microscope. Mu- tants which displayed ER throughout the cell were numer- ous and were discarded since it is well known that ER pro- liferation can be caused by overexpression of membrane proteins or disrupting ER to Golgi complex transport. Three mutants that had disrupted ER structure were iden- tified (WPY152, WPY153, and WPY154). In all three mutants, the mutations that cause the ER structure defects were recessive, segregated 2:2 as deter- mined by back-crosses with a wild-type strain, and were linked to the temperature-sensitive growth phenotype. We isolated plasmids from genomic libraries that comple- mented the growth defect of each of the mutants at 378C. Using a combination of DNA sequencing, subcloning, and integrative mapping, we determined that the mutations re- sponsible for the altered ER structure in the mutants are in SEC27 (WPY153), SRP101 (WPY154), and SRP102 (WPY152). We named the altered alleles of these genes sec27-95, srp101-47, and srp102-510. SEC27 encodes a component of COPI, a coat-forming complex involved in vesicle budding (Duden et al., 1994). SRP101 and SRP102 encode the a and b subunits of the signal recognition par- ticle (SRP) receptor, a heterodimeric complex of GTPases in the ER membrane (Gilmore et al., 1982a,b; Meyer et al., 1982; Ogg et al., 1992, 1998). SRP and SRP receptor target ribosome–nascent polypeptide complexes to the ER membrane for cotranslational protein translocation. We Prinz et al. Mutants Affecting ER Structure 465