additional mutants affecting ER structure that were not identified in the screen. One class of mutants we isolated affects ER structure and the propagation of the ER from the mother to the daughter cell by disrupting vesicular transport between the ER and Golgi complex. Defects later in the secretory pathway did not affect ER structure or inheritance. How exactly vesicular transport between the ER and Golgi complex is required for ER structure is unclear. Resident ER proteins are known to be transported to the Golgi ap- paratus at some low rate (Teasdale and Jackson, 1996), and it therefore seems possible that retrograde transport from the Golgi apparatus to the ER may be required to re- cycle ER proteins needed to maintain proper structure. Surprisingly, we find that ER structure is not affected in a temperature-sensitive mutant with a defect in a protein (Cdc48p) required for homotypic ER fusion in vitro (Lat- terich et al., 1995). Since yeast ER is dynamic, it seems un- likely that ER structure could be maintained in the ab- sence of homotypic ER fusion. It may be that, in vivo, other proteins are able to substitute for the role of Cdc48. Alternatively, it may be that Cdc48p plays a role in the ER–ER fusion required for nuclear division, but is not es- sential for fusion events in peripheral ER. Our data show that the SRP pathway plays an important role in maintaining the reticular structure of the ER. The linkage could be direct. Only mutants with defects in the a and b subunits of the SRP receptor and in SRP itself had the ER disruption phenotype; other mutants with translo- cation defects did not show this effect. In fact, some of the mutations in Sec61p, a component required for transport of all substrates, have stronger translocation defects than the srp101-47 and srp102-510 mutants we isolated (Stirling et al., 1992; Finke et al., 1996; Pilon et al., 1998). In addi- tion, the Sec61p mutants can continue to grow at the non- permissive temperature for a few hours after onset of the translocation defect, and their ER remains normal. De- spite this evidence, it is hard to exclude that the SRP path- way affects ER structure by blocking the translocation of a particular protein. However, we consider it more likely that the SRP pathway affects ER structure via ribosome targeting to the ER membrane. We find a small, but repro- ducible, reduction in ribosome binding to the ER in a SRP receptor mutant, but not a Sec61p mutant or wild-type strain, when they are grown at elevated temperature. In addition, under conditions where ER morphology is restored, ribo- some binding is increased back to wild-type levels. The ex- periments with cycloheximide are consistent with this in- terpretation. Slowing translation with sublethal amounts of cycloheximide restored ribosome binding and sup- pressed both the ER morphology and translocation de- fects of the SRP receptor mutants. These data show that both phenotypes are related to ribosome targeting and ar- gue against an additional function of the SRP pathway. It is unclear how ribosome binding to the ER might affect its structure. In mammals, membrane-bound ribosomes have been proposed to be connected with each other by a pro- tein network (Kriebich et al., 1983; Amar-Costesec et al., 1984; Marcantonio et al., 1984). Perhaps, ribosome binding to the Sec61p or Ssh1p complexes affects this network. A role for ribosome binding in determining ER structure might also explain the morphological differences between the rough and smooth portions of the ER that have been noted since the organelle was first described (Palade, 1956). The SRP pathway is clearly not essential for ER forma- tion and maintenance since cells with deletions of SRP pathway components have normal ER structure. In addi- tion, both the translocation and ER morphology defects in the SRP receptor mutants we isolated are partially amelio- rated during prolonged growth without this pathway. Ad- aptation of SRP pathway mutants has been noted before and was not found to be the result of suppressing muta- tions (Hann and Walter, 1991; Ogg et al., 1992). While it is possible that suppressing mutations may occur in our mu- tants when they are grown at high temperature, it is more likely that they also undergo adaptation. Since we found that ribosome binding is restored under these conditions, our data suggest that it is SRP-independent ribosome tar- geting that restores ER structure. SRP-independent ribo- some targeting may explain both the small changes in ribo- some binding to the ER in SRP pathway mutants when they are shifted to nonpermissive temperature and the fact that the SRP pathway is not essential for viability (Hann and Walter, 1991; Ogg et al., 1992, 1998). Indeed, SRP- independent, direct binding of ribosome–nascent chain complexes to ER membranes has been demonstrated in vitro (Jungnickel and Rapoport, 1995). Finally, our data point to an interesting connection be- tween the structures of the ER and mitochondria. Both organelles form reticular networks close to the plasma membrane and are often closely apposed to one another. A close association between these two organelles has also been observed in mammalian cells (Rizzuto et al., 1998). In SRP receptor mutants, both structures are disrupted, but maintain their association, indicating that they are physically linked at a number of sites. It is possible that the ER is required for maintaining the mitochondrial reticulum. Interestingly, the reverse may not be true since mutations that disrupt mitochondrial structure leave the ER intact. A structural connection between the ER and mitochondria is consistent with known functional interactions between the two organelles. For example, it has been proposed that di- rect contact between the ER and mitochondria is required for phospholipid transport between them (Shiao et al., 1998; Achleitner et al., 1999), and Ca21 released from one or- ganelle can act locally on the other (Rizzuto et al., 1998). We thank R. Schekman, F. Winston, M. Latterich, J. Nunnari, R. Jensen, P. Walter, P. Novick, C. Shamu, D. Botstein, and T. Sommer for strains; C. Shamu, K. Matlack, and L. Dreier for critically reading the manuscript; A. Kram and J. Zagers for help with EM; M. Rolls and J. White for help with quantification of images; and B. Glick for helpful advice. T.A. Rapoport is a Howard Hughes investigator and W.A. Prinz is sup- ported by a postdoctoral fellowship from the American Cancer Society. Submitted: 15 March 2000 Revised: 8 June 2000 Accepted: 12 June 2000 References Achleitner, G., B. Gaigg, A. Krasser, E. Kainersdorfer, S.D. Kohlwein, A. Perktold, G. Zellnig, and G. Daum. 1999. Association between the endoplas- mic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur. J. Biochem. 264:545–553. Amar-Costesec, A., J.A. Todd, and G. Kriebich. 1984. Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum Prinz et al. Mutants Affecting ER Structure 473