The striking cis and trans specificity of Hmg2p stability regulation led us to posit that this process involves a separate set of genes referred to as COD genes. In this work, we have isolated the first member of this class of genes. We focused our search on cod mutants that always degrade Hmg2p even when signals for degradation are low. By our model (), Hmg2p degradation in such a cod mutant would still be halted by mutations in genes encoding the degradation machinery, such as a hrd1 mutant.
The resulting mutant, cod1-1, had the desired phenotype: Hmg2p and Hmg2p-GFP each undergo constitutive degradation that is largely refractory to regulatory signals. Despite the lack of regulation, Hmg2p in the cod1 mutant was degraded at roughly the same rate as in the wild-type under normal growth conditions. Importantly, the constitutively degraded Hmg2p in a cod1 mutant was strongly stabilized by the simultaneous presence of a hrd1 or ubc7 mutant, showing that indeed regulation can be uncoupled from degradation. The cod1 mutant did not globally alter the stability of ER proteins, since cod1 mutation neither destabilized the normally stable Hmg1p-GFP nor altered the degradation rate of the misfolded, constitutively degraded 6myc-Hmg2p-GFP.
The degradation of Hmg2p can be slowed with drugs that block early in the mevalonate pathway or hastened by inhibition of squalene synthase with zaragozic acid (Hampton and Rine 1994
; Hampton and Bhakta 1997
; Gardner and Hampton 1999a
). Both actions arise from alteration of the mevalonate-derived molecule farnesyl pyrophosphate (FPP; Gardner and Hampton 1999a
). The cod1
mutants showed strongly blunted responses to both lovastatin and zaragozic acid. Thus, COD1
is required for coupling the rate of Hmg2p degradation to levels of the FPP-derived signal.
Cod1p is a P-type ATPase. Members of this widely conserved family function in ATP-dependent pumping of ions across biological membranes. The ion specificity of a given P-type ATPase can not yet be determined from sequence information alone. However, our studies indicate that Cod1p may be a Ca2+ transporter. The cod1 phenotype is reversed by addition of Ca2+ to the growth medium of mutant cells, and no other divalent ions tested could do this. Furthermore, treatment of wild-type cells with the Ca2+-preferring chelator EGTA caused aberrant regulation that was similar to the Cod1− phenotype, and specifically reversed by calcium.
This connection between calcium and HMGR regulation is especially intriguing given that regulated degradation of HMGR in mammals is similarly sensitive to perturbations of cellular calcium (Roitelman et al. 1991
; Roitelman and Simoni 1992
). In mammalian cells Ca2+
deprivation specifically inhibits the action of the degradation signal derived from FPP (Roitelman and Simoni 1992
; Meigs et al. 1996
). Similarly, cod1-1
mutants cannot respond to the FPP-derived signal for Hmg2p degradation.
Cod1p appeared to have a fairly specific function. The COD1
gene is not essential and the viable cod1Δ
null mutant has a phenotype identical to that of the cod1-1
allele. A null mutation in COD1
's closest paralogue, YOR291w, had no observable effect on yeast growth or Hmg2p regulation, alone or in combination with the cod1Δ
null. Our ongoing studies have localized the Cod1p protein to the ER (Cronin, S.R., and R.Y. Hampton, manuscript in preparation), and our current model is that Cod1p is a Ca2+
transporter that is important for establishing a lumenal environment appropriate for control of Hmg2p stability. Changes to the ER environment in a cod1
mutant might alter Hmg2p stability by affecting the presentation of the highly specific structural determinants needed for regulated degradation (Gardner and Hampton 1999b
). Alternatively, Cod1p activity may be critical for the function of trans factors regulating Hmg2p degradation.
A model in which Cod1p functions in the ER might explain some of the other phenotypes reported for cod1
was previously identified as SPF1
in an apparently unrelated screen for mutants resistant to a killer toxin. Other phenotypes reported for spf1Δ
null mutants include defective glycosylation of invertase, resistance to vanadate, and sensitivity to hygromycin and calcofluor white (Suzuki and Shimma 1999
). The sensitivity to hygromycin and calcofluor white, indicating defective cell wall synthesis, and the defective glycosylation of invertase all support a role for Cod1p in maintaining the lumenal environment of the ER, an environment thought to be controlled principally by PMR1
The Golgi-localized P-type ATPase Pmr1p is thought to play a major role in maintaining Ca2+
levels in the secretory pathway (Strayle et al. 1999
is required for numerous ER functions and has recently been identified as DER5
, a gene necessary for the degradation of the misfolded lumenal protein CPY* (Duerr et al. 1998
). In contrast to the effect of pmr1Δ
on CPY* degradation, pmr1Δ
had no effect on Hmg2p degradation or its feedback regulation. Thus, Pmr1p and Cod1p appear to have distinct roles, at least in this ER function. There are metazoan P-type ATPase family members of unknown function that have higher similarity to COD1
than to PMR1
. It is tempting to speculate that they may have similar, specialized functions in a variety of organisms.
We currently do not know the mechanism of regulated stability, and one possibility is that there are proteins that specifically protect Hmg2p from degradation when degradation signals are lowered. If such protection factors exist, they would be particularly important both in the basic understanding of regulated ER degradation, and as possible targets for cholesterol lowering drugs. Loss of a protection factor by mutation would cause signal-independent, constitutive degradation of Hmg2p, and so would score as a cod candidate, like cod1-1. However, >300,000 mutagenized colonies of the parent strain were screened yet only alleles (39) of COD1 were recovered. Thus, it may be that the mechanism of Hmg2p regulation does not involve protection factors. Alternatively, it is possible that this version of the COD screen was biased towards recovery of COD1 alleles.
In summary, the above work demonstrates that the genetic approach to understanding regulated degradation of HMGR is a viable one. Integration of the COD1 gene's function into the scenario of Hmg2p regulation and ER function will be an important aspect of completing the picture of HMGR regulated degradation, in yeast and most likely in other eukaryotes as well.