Increased levels of HMG-CoA reductase induce cell type-and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely nonoverlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation.

Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10 (Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7, CUE1, or DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity. Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed coldsensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The sterol metabolite profile of ubc7⌬ cells was altered relative to that of wild-type cells. Since sterol levels are known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was examined at low temperatures. Cells lacking UBC7, CUE1, or DOA10 were cold sensitive, suggesting that these ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis.

Increased expression of certain ER membrane proteins leads to biogenesis of novel ER membrane arrays. These structures provide models in which to explore the mechanisms by which cells control the size and organization of organelles in response to changing physiological demands. In yeast, elevated levels of HMG-CoA reductase induce ER arrays known as karmellae. Cox and co-workers (1997) discovered that karmellae assembly is toxic to ire1 mutants. These mutants are unable to initiate the unfolded protein response, which enables cells to adjust levels of ER chaperones in response to stresses. We sought to determine whether the karmellae-dependent death of ire1 mutants was due to karmellae assembly or to increased levels of HMG-CoA reductase activity. Unexpectedly, we found that ire1 cells could assemble normal levels of karmellae that were structurally identical to those of wild-type cells. In addition, karmellae assembly did not itself induce the unfolded protein response. Certain ire1 strains produced significant numbers of transformants that were unable to utilize galactose as sole carbon source. These results suggest that the karmellaedependent death of certain ire1 strains may simply reflect their inability to grow on galactose.

Increased expression of certain ER membrane proteins leads to biogenesis of novel ER membrane arrays. These structures provide models in which to explore the mechanisms by which cells control the size and organization of organelles in response to changing physiological demands. In yeast, elevated levels of HMG-CoA reductase induce ER arrays known as karmellae. Cox and co-workers (1997) discovered that karmellae assembly is toxic to ire1 mutants. These mutants are unable to initiate the unfolded protein response, which enables cells to adjust levels of ER chaperones in response to stresses. We sought to determine whether the karmellae-dependent death of ire1 mutants was due to karmellae assembly or to increased levels of HMG-CoA reductase activity. Unexpectedly, we found that ire1 cells could assemble normal levels of karmellae that were structurally identical to those of wild-type cells. In addition, karmellae assembly did not itself induce the unfolded protein response. Certain ire1 strains produced significant numbers of transformants that were unable to utilize galactose as sole carbon source. These results suggest that the karmellae-dependent death of certain ire1 strains may simply reflect their inability to grow on galactose.

In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.