Ace1 and Mac1 are known to undergo reciprocal copper metalloregulation (33
). Mac1 is functional as a transcriptional activator in copper-deficient cells, whereas Ace1 is inactive. Cells cultured in standard laboratory culture conditions contain both Ace1 and Mac1 in transcriptionally silent states. Ace1 becomes activated as the cellular copper content is elevated, as seen in copper-supplemented cells. We show presently that cells undergoing a transition from copper-deficient to copper-sufficient conditions through a switch in the growth medium show a dramatic rise in Ace1 activation reported by enhanced CUP1
expression correlates with copper activation of Ace1 (15
). Exposure to copper-deficient conditions results in an up-regulation of the copper uptake system, consisting of the copper permeases Ctr1 and Ctr3 and the Fre1 metalloreductase, necessary to mobilize Cu(I) (33
). Transition of cells in this state to conditions of copper sufficiency results in massive accumulation of cellular copper. Ace1 activation and induction of metallothioneins are likely a protective response to minimize any deleterious effect of the copper shock conditions.
This condition of copper shock resembles a typical situation for microbes in the environment that often experience a shift from nutrient deficiency to repletion. Such transitions result in stress from the nutrient shock (2
). The stress imposed by copper shock can be deleterious, as copper is capable of catalyzing toxic reactions within the cell. The evolutionary pressure for the selection of the Ace1/Cup1 system of copper detoxification may not have been from exposure of cells to high exogenous copper salts in the environment or use in copper culinary vats, but rather from the need to protect cells from copper shock resulting from a change in nutrient availability.
The homeostatic mechanism protecting cells against zinc shock differs from that for copper shock. Cells encountering zinc deficiency up-regulate the plasma membrane zinc uptake transporters and also induce the vacuolar influx zinc transporter Zrc1 (31
). Induction of ZRC1
is a proactive response poising the cell to respond to any subsequent transition to zinc-sufficient conditions in which rapid sequestration of zinc within the yeast vacuole is an important detoxification response. Although copper is sequestered within the yeast vacuole (35
), yeast cells do not appear to induce vacuolar copper sequestration during copper shock conditions.
Cu(I) binding activates the DNA binding function in Ace1 (14
). We confirm that cells undergoing the copper deficiency to sufficiency transition show enhanced Ace1 DNA binding and the corresponding elevation in CUP1
expression. The mechanism of copper inhibition of Mac1 function is more complex. Previous studies suggest copper inhibition of Mac1 to involve attenuated DNA binding and transactivation activity in addition to copper-induced proteolysis. We show presently, using two epitope-tagged variants of Mac1, that copper inhibition is independent of protein turnover. Addition of a TAP tag chromosomally to MAC1
resulted in a chimeric protein level that was increased in copper-replete cells rather than reduced by proteolysis. Likewise, the Myc-tagged chimera consisting of an internal triple Myc tag separating the DNA binding and transactivation domains of Mac1 was also increased, consistent with our earlier study using an overexpressed C-terminal Myc-tagged protein (23
The observed increase in protein levels in copper-replete cells may arise either from copper-induced stabilization of the Mac1 protein or alternatively from reduced stability of the transcriptionally active state (36
). We conclude that copper-induced proteolysis is not the mechanism responsible for copper inhibition of Mac1 function. The observed copper-induced degradation of Mac1 reported previously (43
) may have arisen from copper-dependent removal of the C-terminal hemagglutinin epitope tag, or alternatively Mac1 degradation is only observed in certain genetic backgrounds. It should be pointed out that Mac1 stabilization was observed presently in two distinct genetic backgrounds (W303 and BY4741).
Copper-induced diminution of DNA binding was observed with both epitope-tagged variants of Mac1, consistent with an earlier report (29
). Copper-induced dissociation of Mac1 from target genes is partially responsible for the observed copper inhibition of CTR1
expression. Since chromatin immunoprecipitation analyses reveal only partial Mac1 dissociation, copper inhibition of Mac1 function must also involve copper inhibition of the transactivation activity. We showed previously that transactivation activity in Mac1 is copper regulated (17
) and the transactivation domain is embedded within two Cu(I) binding subdomains (3
Inhibition of the Mac1 transactivation function may arise from direct copper binding or a copper-mediated posttranslational modification. Mutational data on Ace1 and Mac1 are consistent with each protein being a direct copper sensor (21
). Since each protein resides solely within the nucleus, copper metalloregulation may occur by Cu(I) binding to nascent chains emerging from the cytosolic ribosomes or copper translocation to the nucleus. We show presently that copper metalloregulation of Ace1 and Mac1 occurs in cycloheximide-treated cells suggesting that metalloregulation of Ace1 and Mac1 must occur within the nucleus.
Phosphorylation of Mac1 was reported to be essential for DNA binding suggesting that metalloregulation may arise from by a copper-regulated phosphatase (19
). Heredia et al. showed that the addition of calf intestinal phosphatase enhanced the electrophoretic mobility of an epitope-tagged Mac1 (19
). We failed to see any change in the electrophoretic mobility of Mac1 extracted from copper-deficient or copper-supplemented cells. In the absence of identification of a copper-regulated phosphatase in yeast, we focused in the present study on direct copper metalloregulation of Mac1.
One intriguing question arising from copper metalloregulation of Ace1 and Mac1 within the nucleus concerns the pathway of copper shuttling to the nucleus and presentation to the two proteins. Although we have not identified the mechanism of copper delivery to the nucleus, we show for the first time that copper metalloregulation of Ace1 and Mac1 is specific and not altered by high expression of the other copper-binding proteins within the nucleus. High expression of the copper-binding N-terminal domain of Ace1 attenuates the function of endogenous Ace1 but is without effect on the copper inhibition of Mac1 function. Likewise, high expression of a copper-binding, non-DNA-binding Mac1 mutant is without effect on the copper activation of Ace1. Furthermore, the absence of cross-competition and absence of any effect of nuclear Crs5 on copper metalloregulation of Mac1 in transition studies suggest that diffusion of a reactive copper pool such as Cu(I)-GSH is not the source of copper for metalloregulation. A reactive pool of copper exists in cells, as shown by the efficient copper metallation of human Sod1 in yeast cells lacking the Ccs1 metallochaperone (5
Overexpression of CUP1 or CRS5 resulted in enhanced CTR1 expression levels in steady state cultures. Presumably, high levels of Cup1 or Crs5 metallothioneins perturb intracellular copper pools resulting in Mac1 activation. However, overexpression of Crs5 either in the cytoplasm or in the nucleus did not yield the same extent of Mac1 activation as did treatment of cells with the copper chelator BCS. The reason for the greater efficacy of the extracellular BCS chelator compared to intracellular Crs5 in Mac1 activation may relate to the ability of metallothioneins to bind various metal ions. The overexpressed Crs5 may bind metal ions other than copper, thus lowering its ability to deplete copper pools.
No cross competition between Ace1 and Mac1 was observed in cell transition experiments. The lack of cross competition may arise from one of three scenarios. First, if the initial event of Mac1 inhibition is the inhibition of the Mac1 transactivation activity by Cu(I) binding to the C-terminal Cys motifs, the absence of cross competition implies a highly specific route of copper ion presentation to Mac1. This may occur through a specific metallochaperone within the nucleus. Second, copper inhibition of Mac1 function may occur through a signal transduction pathway. Cu(I) binding to Mac1 may be only a late event, perhaps reinforcing the repressed state. High expression levels of Ace1 may not be expected to modulate Mac1 function if signal transduction is the primary inhibitory pathway. Third, the lack of cross competition may arise if Ace1 and Mac1 reside within different subnuclear localizations. The nuclear punctate staining of GFP-Mac1 is clearly distinct from the diffuse nuclear staining of GFP-Ace1.
A rigorous proof of direct copper metalloregulation of Mac1 in cells would require documentation of Cu(I) binding to Mac1 extracted from cells. Due to the low abundance of Mac1, this demonstration has been possible only in cells overexpressing Mac1 (25
). Resolution of this question will require future studies to identify additional proteins that modulate metalloregulation of Mac1.