Elevated expression of Hmg1 in the ubc7Δ, cue1Δ, and doa10Δ ERAD mutants resulted in cold-sensitive growth.
A competitive growth experiment designed to identify genes involved in karmellae biogenesis revealed that ubc7Δ
mutants divided more slowly under karmellae-inducing conditions than other mutants within the population (51
). To confirm these results, ubc7Δ
strains were individually transformed with a plasmid carrying a galactose-inducible HMG1
gene (plasmid AK266) and examined for a galactose-dependent growth phenotype. Both mutants displayed cold-sensitive growth on galactose (Fig. ). Growth was particularly inhibited at 16°C, with moderate inhibition at 26°C. Thus, these ERAD genes appeared to be required for low-temperature viability of yeast cells expressing high levels of HMGR. This result was consistent with the previously observed slow-growth phenotype in the population of deletion mutants, since one of the competitive growth experiments was conducted at 26°C to enable the growth of temperature-sensitive mutants (51
). To determine whether this cold sensitivity was a phenotype limited to the BY4743 strain background, we generated ubc7Δ
mutants in another strain background, JRY527. These new ERAD mutants were similarly cold sensitive in the presence of high levels of Hmg1 (data not shown). Thus, the cold sensitivity was unlikely to result from cryptic mutations or other characteristics unique to the BY4743 background.
FIG. 1. The ERAD proteins Ubc7, Cue1, and Doa10 are required for normal growth in the presence of increased Hmg1. Wild-type, ubc7Δ, cue1Δ, and doa10Δ cells of strain BY4743 were transformed with a plasmid either encoding a galactose-inducible (more ...) Only a subset of genes involved in ERAD or the ubiquitin-proteasome pathway was required for normal growth of cells with elevated levels of Hmg1.
In addition to Ubc7 and Cue1, other ER-associated proteins have a role in ERAD, including Doa10, Ubc6, Hrd1, Hrd3, and Der1 (6
). Based on analysis of growth on solid medium of these mutants, only doa10Δ
mutants exhibited significant cold-sensitive growth in the presence of increased Hmg1 (Fig. and Table ). These results indicated that the ERAD proteins Ubc7, Cue1, and Doa10 were specifically required for normal growth in response to high levels of Hmg1 at low temperatures.
To determine whether the cellular response to increased Hmg1 requires other components of the ubiquitin-proteasome pathway, a panel of deletion mutants were transformed with pAK266 and screened for a growth phenotype at 16, 26, and 37°C (Table ). Many of the mutants showed slight growth inhibition in response to increased Hmg1 at one or more of the temperatures tested. In addition, growth of the doa1Δ mutant expressing elevated HMGR was found to be strongly temperature sensitive. However, none of these mutants exhibited the pronounced cold sensitivity that was observed in ubc7Δ, cue1Δ, or doa10Δ strains.
ubc7Δ, cue1Δ, and doa10Δ cells displayed defective spatial and temporal regulation of karmellae assembly.
Since the Hmg1-dependent growth defects of ubc7Δ
cells were originally recognized as part of a screen to identify genes required for ER membrane biogenesis, we examined whether these mutants were able to assemble normal karmellae. After 24 h of karmellae induction at the nonpermissive temperature of 16°C, ubc7Δ
mutants contained unusual ER membrane proliferations that were not observed in wild-type controls or in previous experiments (50
). In contrast to the highly organized stacks of karmellae membranes that surround the nucleus, forming a horseshoe pattern in sections (Fig. ), the ER membrane proliferations observed in ubc7Δ
were disorganized and were not always closely associated with the nucleus (Fig. ). Thus, although the mutants were able to proliferate their ER in response to elevated Hmg1, they were unable to properly modulate the organization and location of the resulting proliferations.
FIG. 2. ubc7Δ, cue1Δ, and doa10Δ cells make aberrant karmellae. Wild-type, ubc7Δ, and cue1Δ cells of strain BY4743 transformed with pAK266 (carrying a galactose-inducible HMG1 gene) were fixed for electron microscopy after (more ...)
As previously reported, karmellae can be visualized in living cells with fluorescence microscopy using the vital dye, 3,3′-dihexyloxacarbocyanine iodide (DiOC6
]). Subsequent examination of homozygous diploid ubc7Δ
, and doa10Δ
mutants using this method yielded unexpected new information about variation in karmellae morphology in wild-type strains of different backgrounds. All studies of karmellae morphology conducted in our laboratory prior to those presented here have been in yeast strain JRY527. Because ubc7Δ
mutants were identified from a pool of mutants created by the Deletion Consortium, initial investigation of ubc7Δ
mutants and other ubiquitin-proteasome mutants was conducted with mutants derived from the BY4743 parent strain. The wild-type BY4743 cells were difficult to stain with DiOC6
; the cells were very densely stained and appeared to contain some disorganized membrane strips and whorls in addition to normal karmellae. The unusual staining characteristics in this wild-type strain made it difficult to use in vivo observations to determine the extent of karmellae abnormalities in the mutants. Therefore, we examined karmellae structure in ubc7Δ
, and doa10Δ
mutants that were generated in the more easily stained JRY527 background.
As expected from past observations, staining of wild-type JRY527 cells with DiOC6 revealed normal karmellae, without unusual staining observed in the BY4743 background (Fig. ). Consistent with our observations of the ubc7Δ, cue1Δ, and doa10Δ mutants in the BY4743 background, the analogous mutant strains generated in the JRY527 background assembled both karmellae and abnormal membrane structures (Fig. ). Thus, the abnormalities in karmellae assembly in ubc7Δ, cue1Δ, and doa10Δ mutants were unlikely to result from differences in strain background. In the absence of high levels of Hmg1, no unusual membrane structures were observed in any cells examined, although the BY4743 strain continued to be difficult to stain optimally with DiOC6.
Increased HMG-CoA reductase catalytic activity, not karmellae, was responsible for Hmg1 sensitivity in ERAD mutants.
Previous studies have shown that Hmg1-dependent induction of karmellae requires a region in the last ER lumenal region of the membrane domain (“loop G”) and is independent of HMGR catalytic activity (36
). These observations allowed us to test the hypothesis that the cold-sensitive phenotype and abnormal karmellae assembly in ubc7Δ
, and doa10Δ
mutants were functionally related. To determine whether the observed Hmg1-dependent cold sensitivity was due to abnormal karmellae biogenesis, mutants were transformed with plasmids containing mutated or truncated forms of Hmg1 (Fig. ). Wild-type cells transformed with pDP304, which encodes a catalytically inactive form of Hmg1, make normal karmellae when grown on galactose-containing medium but do not express elevated HMGR activity. The ubc7Δ
mutants expressing the catalytically inactive, karmellae-inducing form of Hmg1 (pDP304) grew as well as cells expressing the vector control plasmid. Thus, the catalytic activity was essential for the cold-sensitive phenotype. cue1Δ
mutants transformed with pDP304 also grew similarly to wild-type (data not shown.)
FIG. 3. HMG-CoA reductase catalytic activity, not the presence of karmellae, is the cause of decreased growth in response to increased levels of Hmg1. (A) Wild-type and ubc7Δ cells were transformed with vector control or plasmids expressing galactose-inducible (more ...) ubc7Δ
cells expressing a galactose-inducible, catalytically inactive form of Hmg1 that is unable to induce karmellae (p260) grew as well on galactose-containing media as cells transformed with pDP304. Conversely, ubc7Δ
cells expressing a galactose-inducible mutant form of hmg1
that was unable to induce the formation of karmellae but retained catalytic activity (p558) was cold sensitive. ubc7Δ
mutants constitutively expressing HMG1
under the control of the GPDH
promoter (p716) showed a slight cold-sensitive phenotype on both glucose and galactose at 16°C, indicating that the observed phenotype is not carbon source dependent (Fig. ). Finally, ubc7Δ
cells expressing galactose-inducible Hmg2 (pRH134-2) grew more poorly on galactose than vector control transformants. The Hmg2 protein has identical catalytic activity as Hmg1 but induces proliferation of short stacks of smooth membranes that can be associated with the nucleus or plasma membrane or present in the cytoplasm (28
). Collectively, these observations indicate that increased HMGR activity, not abnormal karmellae assembly, was responsible for the cold-sensitive phenotype.
To confirm that the primary cause of Hmg1-dependent cold sensitivity in ubc7Δ, cue1Δ, and doa10Δ cells was HMGR catalytic activity, mutant cells expressing pAK266 were grown at 16°C in the presence of lovastatin, a competitive inhibitor of HMGR. As previously observed, the mutants expressing high levels of Hmg1 displayed cold-sensitive growth when they expressed elevated HMGR. However, when HMGR activity was reduced by the presence of lovastatin, nearly normal growth was restored at 16°C (Fig. .) This result demonstrated that the increased catalytic activity of Hmg1 is toxic at cold temperatures to ubc7Δ, cue1Δ, and doa10Δ mutant cells.
The response of proteasome mutants to increased Hmg1 was similar to that of ubc7Δ, cue1Δ, and doa10Δ cells.
Because Ubc7, Cue1, and Doa10 are part of the molecular machinery that covalently attaches ubiquitin to target proteins, we hypothesized that the Hmg1-dependent cold sensitivity in these mutants was due to the failure of these mutants to ubiquitinate a specific target protein or proteins. Ubiquitination of this target might result in either activation of a proteasome-independent event (41
) or degradation of the protein by the proteasome (29
). To distinguish between these two possibilities, we examined the growth characteristics of proteasome mutants that expressed increased levels of Hmg1. If an inability to degrade the target protein were the basis for the Hmg1 sensitivity, then cells with defects in proteasome function should show similar cold-sensitive phenotypes as those observed in ubc7Δ
, and doa10Δ
Although genes encoding proteins that compromise the proteasome are essential, partial-loss-of-function mutants are viable. Strain WCG4/11-12 (pre1-1 pre1-2
) exhibits partial loss of function of two essential 20s core particle components, Pre1 and Pre2, and has been shown to have 5% of normal proteasome activity (18
). The growth of this mutant strain and a congenic wild-type expressing elevated Hmg1 was examined.
Under all conditions tested, pre1-1 pre2-1 mutants grew less well than the wild type. In addition, mutant transformants displayed variability in growth that was not observed in the congenic wild-type transformants. Consequently, to ensure that conclusions about the effects of increased levels of Hmg1 were not confounded by variability in growth of independent transformants, 29 randomly selected pre1-1 pre2-1 vector control transformants and 29 randomly selected pAK266 transformants were examined. Two representative transformants for each plasmid are shown in Fig. . As expected, all 29 of the pre1-1 pre2-1 vector control transformants that were examined grew as well with normal levels of Hmg1 (i.e., on glucose) as with high levels of Hmg1 (i.e., on galactose). Of the 29 pre1-1 pre2-1 mutants transformed with AK266, 25 exhibited Hmg1-induced cold sensitivity, a finding consistent with the hypothesis that normal growth of cells with elevated levels of HMGR requires ubiquitin-mediated protein degradation. Given the poor growth of pre1-1 pre2-1 mutants, the four transformants with normal growth may have gained reversion or suppressor mutations that elevate proteasomal function. Interestingly, the Hmg1-induced growth inhibition observed in proteasome mutants was not as extreme as that observed in ERAD mutants. Therefore, the cellular response to increased Hmg1 may require additional ERAD-dependent events that are proteasome independent.
FIG. 4. Proteasome function mutants are sensitive to increased levels of Hmg1. A pre1-1, pre2-1 mutant strain and a congenic wild-type strain were transformed with either pAK266 or vector control plasmids. The left-most spot is inoculated from a culture at 1.4 (more ...) Deletion of HMG2 did not suppress cold sensitivity in ubc7Δ and cue1Δ cells.
The cold sensitivity of the pre1-1 pre2-1
strain described above suggests that one component of the normal cellular response to increased levels of Hmg1 is the UBC7-
, and DOA10-
dependent degradation of a protein target. Hmg2 is a known ER-resident target of Ubc7 and the E3 ubiquitin ligase, Hrd1 (11
). Although Doa10 and Hrd1 have been shown to have distinct substrate specificity, experimental evidence suggests that Hrd1 and Doa10 have some overlapping function (45
). Therefore, we hypothesized that under our unique experimental conditions, the inability of the ubc7Δ
, and doa10Δ
mutants to degrade Hmg2 might be the underlying molecular cause of their sensitivity to elevated Hmg1 expression. If so, deletion of HMG2
in any of the mutant strains should suppress the cold-sensitive phenotype. Double-deletion mutants lacking both UBC7
were generated in the JRY527 background and transformed with either pAK266 or a vector control plasmid. As seen in Fig. , deletion of HMG2
did not restore the growth observed in vector controls to ubc7Δ
cells and, therefore, did not suppress the Hmg1-induced cold sensitivity. Therefore, Hmg2 is unlikely to be the essential target of UBC7-
, and DOA10-
FIG. 5. Deletion of HMG2 does not suppress cold sensitivity in ubc7Δ cells. A ubc7Δ hmg2Δ double deletion mutant strain in the JRY527 background was transformed with either pAK266 or vector control plasmids. The left-most spot was inoculated (more ...)
Although Hmg1 is not a substrate for ERAD under standard growth conditions (11
), we hypothesized that it might become subject to degradation under certain conditions, such as growth at low temperatures. To test this hypothesis, total protein was isolated from wild-type, ubc7Δ
, and doa10Δ
cells transformed with pAK266 after 24 h of karmellae induction at permissive and nonpermissive temperatures. Immunoblot analysis with an antibody specific to the Hmg1 isozyme revealed that total levels of Hmg1 in mutant cells were the same as or lower than that of wild-type cells (data not shown). Thus, the loss of these ERAD proteins did not lead to the elevation of Hmg1 levels, making it unlikely that Hmg1 itself is the essential target of Ubc7, Cue1, and Doa10.
Sterol metabolite profiles were altered in ubc7Δ cells.
HMGR catalyzes the formation of mevalonate, the rate-limiting step in the biosynthesis of sterols and other isoprenoids in animals and fungi (12
). A reasonable molecular mechanism for the Hmg1 sensitivity observed in mutant cells is that increased flux through the sterol biosynthetic pathway results in accumulation of a toxic metabolite whose levels are normally held in check via the action of Ubc7, Cue1, and Doa10. For example, Donald et al. showed that cells overexpressing the Hmg1 catalytic domain accumulate increased squalene levels and show decreased growth rates (11
GC was used to analyze sterol metabolite composition of ubc7Δ and wild-type cells in the BY4743 background in the presence of normal and increased levels of Hmg1 (Fig. ). Although this method did not measure absolute sterol levels, it provided quantitative data concerning the relative amounts of particular sterols within a sample. Interestingly, increased levels of Hmg1 resulted in a decrease in the percentage of ergosterol in both wild-type and ubc7Δ cells at both permissive and restrictive temperatures. This result suggested that one or more sterol biosynthetic enzymes that catalyze reactions downstream of squalene synthase were downregulated in response to elevated flux through the sterol biosynthetic pathway. In addition, this regulation appeared to be intact in ubc7Δ mutants.
FIG. 6. The sterol metabolite profile of ubc7Δ cells in the presence of normal and increased levels of Hmg1 differs from the isogenic wild-type control. GC was used to measure the relative amounts of sterol metabolites as a percentage of total cellular (more ...)
The proportion of squalene in wild-type cells expressing elevated levels of Hmg1 was higher than in the vector control. However, the proportion of squalene in ubc7Δ mutants was actually lower than that of wild-type cells. The observation that ubc7Δ cells exhibited lower squalene levels ruled out the possibility that accumulation of excess squalene in ubc7Δ cells was responsible for their Hmg1 sensitivity.
Although the proportion of squalene was lower in ubc7Δ
cells than in the wild type, the proportion of several other sterol metabolites was elevated in ubc7Δ
cells grown at 16°C with high levels of Hmg1. If the Ubc7/Cue1/Doa10 complex regulates flux through the sterol biosynthetic pathway by targeting ergosterol biosynthetic enzymes for degradation, then the loss of UBC7
function should lead to elevated levels of these enzymes, in turn producing inappropriately elevated amounts of their products. Thus, the elevated proportions of lanosterol (synthesized by Erg7), 4,4-dimethylzymosterol (synthesized by Erg24), fecosterol (synthesized by Erg6), and 4-methylfecosterol (resulting from incomplete C-4 demethylation by the Erg25, Erg26, and Erg27 complex) in ubc7Δ
mutants suggest that the essential substrate(s) of the Ubc7/Cue1/Doa10 complex may be Erg6, Erg7, Erg24, Erg25, Erg26, and/or Erg27. Of these potential target proteins, only Erg6 and Erg24 are nonessential. Erg6 is a soluble protein that is associated with lipid particles and the ER (2
). Erg24 is an ER transmembrane protein (30
). Thus, the localization of both proteins is consistent with the possibility that they are ERAD targets. We constructed ubc7Δ erg6Δ
and ubc7Δ erg24Δ
double mutants to test directly whether loss of these erg
genes suppressed the cold-sensitive phenotype of ubc7Δ
mutants. In both cases, the double mutants were as cold sensitive as the ubc7Δ
mutants alone (data not shown.) Thus, neither the loss of Erg6 nor the loss of Erg24 individually suppressed the cold sensitivity of ubc7Δ
ubc7Δ, cue1Δ, and doa10Δ mutants are cold sensitive in the absence of elevated levels of HMGR.
The results presented thus far suggested that the Ubc7-Doa10 ERAD pathway is important for regulating sterol biosynthesis. In addition, it appears that this regulation is particularly important at low temperatures. Interestingly, Fig. shows that expression of increased levels of HMGR in wild-type cells growing at 30°C results in sterol profile changes that are similar to those seen in wild-type cells growing with normal levels of HMGR at 16°C. In addition, the sterol profiles of ubc7Δ mutants were abnormal under all conditions tested. Given these results, we hypothesized that increased levels of HMGR produce cellular responses similar to low temperature growth. According to this scenario, cells growing at 16°C with high HMGR levels would respond physiologically as if they were growing at a lower temperature. To begin exploring this hypothesis, we examined the ability of ubc7Δ, cue1Δ, and doa10Δ mutants expressing normal levels of HMGR to grow at 10°C. As predicted, these ERAD mutants, but not others, are cold sensitive, although ubc7Δ and cue1Δ mutants are more cold sensitive than the doa10Δ mutant (Fig. .)
FIG. 7. The ERAD proteins Ubc7, Cue1, and Doa10 are required for normal growth at low temperature in the absence of high levels of Hmg1. Wild-type yeast strains and cue1Δ, doa10Δ, and ubc7Δ mutants were serially diluted (1:5) and plated (more ...)