Mutants Affecting the Structure of the Cortical Endoplasmic Reticulum in Saccharomyces cerevisiae William A. Prinz,* Lara Grzyb,* Marten Veenhuis,§ Jason A. Kahana,‡ Pamela A. Silver,‡ and Tom A. Rapoport* *Department of Cell Biology and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115; ‡Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School Dana Farber Cancer Institute, Boston, Massachusetts 02115; and §Laboratory for Eukaryotic Microbiology, GBB, University of Groningen, 9750 AA Haren, The Netherlands Abstract. We ﬁnd that the peripheral ER in Saccharo- myces cerevisiae forms a dynamic network of intercon- necting membrane tubules throughout the cell cycle, similar to the ER in higher eukaryotes. Maintenance of this network does not require microtubule or actin ﬁla- ments, but its dynamic behavior is largely dependent on the actin cytoskeleton. We isolated three conditional mutants that disrupt peripheral ER structure. One has a mutation in a component of the COPI coat complex, which is required for vesicle budding. This mutant has a partial defect in ER segregation into daughter cells and disorganized ER in mother cells. A similar phenotype was found in other mutants with defects in vesicular trafﬁcking between ER and Golgi complex, but not in mutants blocked at later steps in the secretory pathway. The other two mutants found in the screen have defects in the signal recognition particle (SRP) receptor. This Introduction The ER usually consists of a polygonal meshwork of mem- brane tubules and sheet-like cisternae (Palade, 1956). These structures are connected with one another and with the continuous membrane of the outer nuclear envelope. Much of the ER, called RER, is involved in protein trans- location. It is studded with ribosomes carrying nascent chains that are transferred into or across the ER mem- brane as they are being synthesized. In some cells, smooth regions of the ER are found that lack ribosomes and have a different structure and function. Little is known about how the complex morphology of the ER is formed and Address correspondence to Tom A. Rapoport, Department of Cell Biol- ogy and Howard Hughes Medical Institute, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: (617) 432-0367. Fax: (617) 432- 1190. E-mail: tom_rapoport@hms.harvard.edu Jason A. Kahana’s current address is Ludwig Institute for Cancer Re- search, CMM-East, Rm 3080, 9500 Gilman Dr., La Jolla, CA 92093-0660. receptor, along with SRP, targets ribosome–nascent chain complexes to the ER membrane for protein translocation. A conditional mutation in SRP also dis- rupts ER structure, but other mutants with transloca- tion defects do not. We also demonstrate that, both in wild-type and mutant cells, the ER and mitochondria partially coalign, and that mutations that disrupt ER structure also affect mitochondrial structure. Our data suggest that both trafﬁcking between the ER and Golgi complex and ribosome targeting are important for maintaining ER structure, and that proper ER struc- ture may be required to maintain mitochondrial structure. Key words: SRP receptor • endoplasmic reticulum • cytoskeleton • mitochondria • yeast maintained, or what role the structure plays in functions of the ER. In mammalian cells, the ER tubule network is highly dy- namic and is continually being extended to the periphery of the cell (Lee and Chen, 1988; Waterman-Storer and Salmon, 1998). It is rearranged by tubules sliding along one another and by new tubules emerging from preexist- ing tubules and fusing with others. The formation and maintenance of the ER network is thought to require mi- crotubules (Terasaki et al., 1986; Terasaki, 1990). Depoly- merization of microtubules with drugs like nocodazole causes a gradual and reversible contraction of the ER to the center of the cell (Lee et al., 1989). Newly forming tu- bules are thought to be pulled out of existing tubules by motor proteins migrating along microtubules and/or by being dragged by the tip of polymerizing microtubules. In vitro, microtubules have also been proposed to be re- quired for the formation of ER-like networks of mem- brane tubules (Dabora and Sheetz, 1988). However, the in ª The Rockefeller University Press, 0021-9525/2000/08/461/14 $5.00 The Journal of Cell Biology, Volume 150, Number 3, August 7, 2000 461–474 http://www.jcb.org 461