In the present study, we report the first response to heterologous protein production at the translational level. We demonstrated that heterologous expression of soluble or integral membrane proteins or overexpression of an endogenous membrane protein derepresses translation of GCN4
mRNA. Heterologous expression of the Na,K-ATPase integral membrane protein induced GCN4
translation between 4- and 70-fold compared to the control, depending on the strain background and the expression system. The soluble part of the Na,K-ATPase, α1
(TM4/TM5), only induced GCN4
translation in the S288C mutant background. This is in accordance with results obtained for amino acid starvation, which caused a strain-dependent increase in GCN4
). The presented observations widen the spectrum of stress responses involving the Gcn4p transcription factor to include a response of considerable importance for basic and applied sciences.
A reduction in the rate of translation initiation is a well-characterized stress response in mammalian cells which is directed against viral infections or an unfavorable folding environment in the ER. These conditions reduce translation initiation in general through phosphorylation of eIF-2α. In yeast, however, eIF-2α phosphorylation caused by amino acid starvation does not substantially inhibit general protein synthesis but, rather, increases the translation of a subset of mRNA species (16
). In accordance with this, heterologous expression also does not affect translation initiation within a time span where other stress situations such as glucose depletion and osmotic shock decrease translation initiation, as the polysome distribution was unaffected by heterologous expression. Our data also indicate that transcription in general is unaffected by heterologous expression, as the density of the short-lived PAB1
mRNA was grossly unaltered for 72 h after the induction of recombinant protein production. Our results therefore exclude overall reduced macromolecular synthesis as the cause of low heterologous membrane protein production.
Besides amino acid starvation, Gcn4p biosynthesis is induced by starvation for purines (31
), glucose limitation (41
), growth on ethanol (41
), high salinity in the growth medium (8
), treatment with methyl methanesulfonate (31
), or treatment with the Tor1p and Tor2p inhibitor rapamycin (36
). In these cases, translational induction of GCN4
is part of the cellular response to changes in the environment. However, our results demonstrate that internal stress factors like heterologous expression or endogenous membrane protein overexpression also induce translational induction of GCN4
. This underscores the point made by Natarajan et al. (27
) that Gcn4p is a master regulator of gene expression able to induce the transcription of a number of genes in response to a panoply of stress situations.
The induction of GCN4
translation usually requires the Gcn2p kinase (17
). Heterologous expression also induces GCN4
translation in a Gcn2p-dependent manner, as translational induction was not observed in a gcn2Δ
The only known substrate for Gcn2p is eIF-2α, and all Gcn2p-dependent stress responses, except for those in response to UV irradiation (7), involve eIF-2α phosphorylation. This is also the case for heterologous expression, as derepression of GCN4 mRNA translation required serine 51 in eIF-2α and was accompanied by an increase in eIF-2α phosphorylation that was sustained for at least 72 h after the induction of recombinant protein production.
The only known way to activate Gcn2p is through binding of uncharged tRNA to its histidyl-tRNA-like domain. This domain is also essential for the derepression of GCN4 mRNA translation in response to heterologous expression, as the gcn2m2 allele prevented Gcn4p from accumulating to a high level.
The Gcn1p-Gcn20p complex that is essential for the activation of Gcn2p through interactions with its amino terminus was also found to be required for the stimulation of Gcn2p in the present study. It has been suggested that the ribosome-associated Gcn1p-Gcn20p complex may either facilitate binding of uncharged tRNA to the ribosomal A site or facilitate transfer from the A site to Gcn2p (40
). Since translational derepression of GCN4
due to heterologous expression was shown to rely on the tRNA binding domain in Gcn2p, it could be speculated that heterologous expression of cDNA containing rare codons would increase the ratio between the relevant uncharged tRNA and charged tRNA and consequently stimulate Gcn2p activity. However, the expression of the Na,K-ATPase from a chromosomal copy of the expression plasmid derepressed GCN4
translation to the same extent as expression from a very-high-copy-number plasmid, although the density of Na,K-ATPase molecules in yeast membranes only reached 1‰. It would, however, be hard to imagine that such a low expression level would drain the cell of charged tRNA molecules specific for rare codons. At least in Escherichia coli
, rare codons only seem to be prohibitive for very high expression levels (22
The endogenous H-ATPase Pma1p is the most abundant plasma membrane protein in yeast, accounting for up to 25% of the total plasma membrane protein content (32
). Therefore, yeast does have the capacity to deposit at least one membrane protein in the plasma membrane at a high density. This density of Pma1p is very high compared to the density of about 1% for recombinant Na,K-ATPase in the plasma membrane of yeast (29
). The fact that a density of as little as 1‰ for Na,K-ATPase induced GCN4
translation as efficiently as 1%, while an expression level of 25% for Pma1p did not trigger the GCN4
stress response, illustrates the sensitivity of the system and its ability to discriminate between endogenous and heterologous protein production. The GCN4
system does, however, respond to a two- to threefold overexpression of the endogenous Pma1p protein. In view of the initially high Pma1p content, this represents a huge density of Pma1p in yeast membranes, demonstrating that yeast has the capacity to express huge amounts of membrane protein. These data point to a new role of GCN4
in responding to unbalanced expression of endogenous proteins.
To determine what step in the biosynthesis of membrane proteins was monitored by the GCN4
system, we took advantage of previously characterized temperature-sensitive Na,K-ATPase mutants. In contrast to wild-type Na,K-ATPase, these mutant proteins only accumulated to a wild-type level at 15°C, and not at 35°C, due to misfolding in the ER lumen, as determined by induction of the unfolded protein response. The mutant proteins were synthesized at rates comparable to that of the wild type at both temperatures (21
). The observation that the expression of wild-type and mutant Na,K-ATPases induced GCN4
translation to the same extent irrespective of the temperature excludes the possibility that Gcn4p is synthesized in response to the presence of a single class of misfolded protein in the ER lumen. This scenario would also contradict the observation that expression of the cytoplasmic α1
(TM4/TM5) loop protein induces GCN4
translation without postulating the existence of a cytoplasmic sensor. This was confirmed by the fact that interference with general protein folding in the ER lumen only induced the unfolded protein response and not GCN4
mRNA translation. Thus, the induction of GCN4
translation by heterologous expression is unrelated to the ability of the protein to fold in the ER lumen. Consequently, the experimental data indicate that a step in heterologous protein production or endogenous protein overexpression preceding entry into the ER lumen must activate Gcn2p and cause eIF-2α phosphorylation.
The Na,K-ATPase is responsible for maintaining Na+
gradients across the plasma membrane in all higher eukaryotes except for plants. Even though the Na+
concentration in yeast minimal medium is very low, in situ pump activity might interfere with ion homeostasis and cause an induction of GCN4
translation. This can be ruled out, however, as expression of an enzymatically inactive Na,K-ATPase (30
) induced GCN4
translation to the same extent as the wild type. Imbalanced Na+
gradients are therefore not the cause of GCN4
Translational induction of GCN4 seems to be a general response to at least heterologous membrane protein production, as the 7TM receptor PACR1 induced translation to the same extent as the Na,K-ATPase.
What are the promoter targets for Gcn4p produced in response to heterologous expression? A classical Gcn4p target is the HIS4
promoter, which only relies on this transcription factor for activity (24
). It was therefore surprising, to some extent, that the increased Gcn4p concentration was not followed by increased HIS4
transcription. On the contrary, HIS4
transcription was reduced after the induction of heterologous membrane protein production. This parallels the observations made under glucose starvation, which was shown to increase GCN4
translation but reduce HIS4
mRNA levels to near the detection limit (41
). This indicates that the amount of Gcn4p protein cannot be taken as an indicator of Gcn4p transcriptional activity and that Gcn4p may be modified in response to the stimulus that triggered its biosynthesis. This modification might prevent localization to the nucleus or interactions with the basal transcriptional apparatus. Mbf1p is required for the induction of classical promoters targeted by Gcn4p (34
). The observation that the classical HIS4
promoter was not activated in response to heterologous expression despite increased production of Gcn4p may indicate that interactions with Mbf1p at this promoter are prevented during heterologous expression. Posttranslational modifications could be a way to destroy Gcn4p-Mbf1p interactions and allow interactions with other, presently unknown, promoters.
The classical way to induce GCN4 translation is starvation for amino acids, but glucose starvation also increases GCN4 translation. Induction by heterologous expression might therefore be due to a higher rate of amino acid and/or glucose consumption in yeast expressing the heterologous protein than in the control. If this were the case, one would not expect a constant low level of GCN4 translation in the control strain over the entire 72 h of the experiment but rather a later onset of GCN4 translation than that in the Na,K-ATPase-expressing strain. Also, experiments performed in the presence of either 0.5% or 2% glucose or in the presence or absence of amino acids in the growth medium ruled out the possibility that starvation for amino acids or glucose is responsible for the observed induction of GCN4 translation. A further argument for this comes from the fact that growth in raffinose as the sole carbon source showed the same induction of GCN4 translation as growth in 0.5 or 2.0% glucose.
Since uncharged tRNA is the compound activating Gcn2p kinase, the induction of GCN4
translation by heterologous expression could be caused by reduced cytoplasmic levels of amino acids, as observed in the case of glucose limitation (41
). Reductions in cytoplasmic levels of amino acids cannot be due to auxotrophy, as all yeast strains carrying expression plasmids or empty control vector used in the present study are prototrophs. Determinations of the cytoplasmic and vacuolar amino acid pools excluded the possibility that the observed induction of GCN4
translation was related to pool size, as these were similar for the heterologously expressing strain and the control. Thus, conditions previously described to induce GCN4
translation seem unable to explain the presently observed increase in GCN4
translation, although it cannot be ruled out by our experiments that the concentration of a single amino acid could have been limited. This is not likely, however, as these experiments were performed in the presence of amino acids and even the exclusion of amino acids from the growth medium did not evoke translational induction of GCN4