In this study, we have identified biological mechanisms related to protein synthesis and secretion by introducing perturbations to the cell, in the form of HAC1 deletion and different recombinant protein expression, and measuring the system level cellular responses, via transcriptomics and metabolic fluxes. These measurements, combined with data analysis algorithms, Reporter TF algorithm and FBA, were able to identify cellular adjustments in (a) overall expression level, (b) post-Golgi sorting, (c) amino acid biosynthesis and savaging, and (d) oxidative stress. These biological effects are a result of the combined influence of protein synthesis and trafficking through the secretory pathway.
Overall transcription and translation were repressed in response to α-amylase expression (a larger protein) and in the Δhac1 strains with any recombinant protein secretion. Repressing overall expression is a broad spectrum response used to adjust the rates of all other cellular processes to match the reduced folding capacity in the ER. Several mechanisms were used to alter overall expression: repressing mRNA synthesis, increasing mRNA degradation rates, and repressing protein translation rates through reducing ribosome numbers. Specifically, mRNA concentrations are lowered by decreasing RNA polymerase accessibility (HIR2), inhibiting transcriptional elongation (THO2), and controlling RNA degradation (STO1) [31, 32]. Ribosome concentration, and thereby translation rates, can be reduced by the TFs Fhl1p and Rap1p which control expression of rRNA and ribosomal proteins . This is seen in IP production in Δhac1 strain, both by the reporter TFs (Figure 3) and by expression of ribosomal proteins (Additional file 7). In this context, extrachromosomal plasmids offer advantages over chromosomal expression. HIR2, whose mechanism is to silence the chromosome, would not affect extrachromosomal plasmids. Increased recombinant protein secretion would be accomplished by silencing native ER genes, while recombinant, plasmid-born gene would not be affected.
Pronounced adjustments to the TGN were observed in the transcriptome in all conditions. TFs involved in pheromone responses (STE12, MCM1, ASH1), invasive/pseudohyphal growth (STE12, MSN1, PHD1, RIM101), and osmotic stress (CIN5, SKN7, SKO1, YAP6, MSN1) were all identified by the Reporter TF algorithm and point to an underlying set of activities that are required to increase the traffic of secretory vesicles to the membrane. Invasive, pseudohyphal, and filamentous growth morphologies have a high surface to volume ratio and inherently require higher Golgi-to-cell membrane trafficking rates to supply cell membrane and cell wall components for growth. These altered morphologies can be activated through the filamentous and invasive response elements (FREs)  bound by STE12 and used to regulate PHD1 . HAC1 deletion has been shown to cause filamentous growth .
Osmotic stress TFs are also responsible for affecting protein secretion, as the external cell wall must be strengthened in response to hypo-osmotic conditions, thereby requiring an efficient secretory pathway to ferry cell wall proteins . MSN1 is known to induce starch degradation, requiring the actions necessary to secrete the appropriate enzymes through filamentous growth activation . SKN7 has a dual role in invasive growth and osmotic stress . Although osmotic stress TFs are commonly associated with the hyper-osmotic glycerol (HOG) pathway, Ypd1p can phosphorylate Skn7p, signaling the hypo-osmotic stress pathway . Because there were no apparent hypo-osmotic conditions in this study, this indicates that these TFs are not directly controlled by osmotic conditions, but possibly through a secondary response to upregulation and increased secretion of cell wall proteins.
TGN TFs and/or the genes they regulate are possible targets for increasing Golgi-to-cell membrane trafficking. In S. cerevisiae, recombinant protein intended for secretion has been found mis-trafficked to the vacuole. This has been shown for insulin and green fluorescent protein secretion in yeast [40, 41]. Proteins involved in vesicle trafficking, namely Sly1 and Munc18 have been found to increase recombinant secretory rates in Chinese hamster ovarian (CHO) and several mammalian cell lines [42, 43]. It is likely that similar proteins are present in yeast and could be exploited for improving protein secretion.
Significant alterations in amino acid metabolism were observed, particularly in the Δhac1 strains. De novo amino acid synthesis (GCN4, BAS1, MET32, ARG81, RTG3) was suppressed. On the surface, this appears contradictory, as increased amino acid requirements should be observed with recombinant protein production. However, this decrease in de novo amino acid synthesis is accompanied by observed increases in scavenging mechanisms for amino acids (SNT2, CUP9, PUT3). High scavenging rates and decrease synthesis imply high protein degradation rates where the degraded proteins result in available amino acids for scavenging; reducing the need for newly synthesized amino acids. This is consistent with either ERAD, a process where proteins that are stalled in the ER are transported back into the cytoplasm for degradation by the proteosome, or vacuolar-localized protein degradation. In either case, the cell is expending energy on synthesizing proteins that are ultimately degraded. These effects appear in the strains that are the slowest growing with the highest ATP requirements (Figures 1b and 2b). In these cases the ER folding capacity is likely saturated, resulting in ER holdup and ERAD.
Oxidative stress TFs were also found in all conditions. Several were dual oxidative/osmotic stress TFs (CIN5, SKN7, SKO1), and others were dedicated to oxidative stress only (AFT2, YAP1). TFs were found in all three of the major oxidative stress signaling pathways, (a) the Hog1 MAPK pathway (where SKO1 is the DNA binding agent), (b) Sln1 pathway (where SKN7 is the DNA binding agent), and (c) YAP1 and CIN5, which directly sense oxidative stress and bind DNA . The cell's control machinery appears to have hard-wired oxidative stress responses to increased secretory demand, as oxidative/hypo-osmotic pathways have a high degree of overlap, which is appropriate because increased secretion of cell wall proteins will result in higher oxidative stress. In particular, Skn7p, which has already been mentioned for its role in managing secretory pathway directly in an osmotic stress pathway, can also activate oxidative stress response genes .
Oxidative stress was pronounced with all secretory perturbations and has been identified in other studies to be associated with secretory stress [1, 17]. Futile cycling may be the dominant disulfide resorting pathway when folding is limited. In previous studies, oxidative stress, induced by tunicamycin, a N-linked glycosylation inhibitor, increased with ER stress, despite no increase in the net disulfide bond formation demand . The futile cycle does predict non-stoichiometric ROS formation, while isomerization does not. ROS can be formed at potentially limitless amounts through multiple rounds of disulfide formation and breaking. This will occur under conditions where the rate of folding is slow, a result of proteins that are specifically difficult to fold, or a result of the overall ER folding capacity being saturated. As well, futile cycling will increase as the number of available cysteine residues available for disulfide bonding increase, as is the case for α-amylase, due to the extended amount of isomerization that may be needed to form the correct disulfide bonds.
One implication of the proposed thermodynamic model is that PDI paralogues, or cysteines within a PDI, must exist at different electron affinities that are above and below the electron affinity of the protein to be folded. Although in vivo redox potentials of PDI cysteine pairs were not measured, from first principles it would appear highly likely that these PDIs would need different redox potentials to carry out isomerization. In Figure 4a, we assume that only PDIs interact with the folding protein. This appears the case, as kinetic rates for direct glutathione oxidation/reduction are too slow to be physiologically relevant . Electron affinity (and therefore redox potential) is broadly determined by the proximity of the two cysteines, with the proximity determined by the current structure of the protein . Cysteines that are in the correct orientation will have a low electron affinity and easily form disulfide bonds, while cysteines that are not in the correct orientation will have a high electron affinity and will have unstable disulfide bonds. Therefore, the electron affinity of a correctly folded/correct disulfide bond would be lower than that of a misfolded or incorrect disulfide bond. This difference in electron affinity may allow PDIs to selectively break disulfides with high electron affinity (incorrect bonds), but not disulfide bonds with low affinity (correct bonds).
The need for different PDIs to form or break disulfide bonds may explain the need for many PDI homologues in the ER, each with different structures, and therefore different electron affinities. These PDIs can only span a finite range of electron affinities, and there may be implications for proteins that have disulfide pairs with electron affinities higher than the highest PDI or lower than the lowest PDI. If no PDI has a lower electron affinity than an incorrect disulfide bond, then the disulfide bond cannot be broken and the protein is terminally misfolded. As well, a protein that has a native disulfide pairing with an electron affinity higher than any PDI cannot form a bond. This may be the case when recombinant proteins are being processed in the ER.
Futile cycling as a large potential ROS source has broad implications on the cell. Tu and Weissman predict Ero1p-produced ROS that is one-to-one with disulfide bond formation could attribute approximately 25% of cellular ROS to the secretory pathway . Therefore, even larger ROS production is likely if the futile cycle is the dominant disulfide resorting pathway under folding stress. This also has implications on GSH and possibly NADPH availability, as it is doubly consumed (a) by the reduction of ROS and (b) directly in the futile cycle. The futile cycle limits reducing equivalents needed for anabolic processes, and may explain the reduced growth rates observed in folding stressed strains (WA, dI, and dA).
In all, Figure 4b highlights that the relative rates of two processes, protein folding and disulfide bond formation, must be kept in balance to avoid significant cellular stress. If disulfide bond formation is fast compared to folding, high futile cycle use will result in high ROS formation, NADPH loss, and high protein degradation as a result of ERAD. This scenario is observed in the Δhac1 strains dI and dA.
The engineering implications for protein secretion become much clearer with this understanding of protein folding to disulfide bond formation ratio. When overexpressing a recombinant protein, an optimal expression must be found, where transcription is as high as possible without overloading the ER folding capacity and sending the cell into an oxidative stressed state. This optimal expression level will be different for different proteins, as protein folding rates will vary according to the protein size and structure. We see this in comparing IP and α-amylase expression. The concept of an optimal expression has been identified heuristically, in the present study we identify the competing molecular effects that could define these phenomena . This optimal expression ratio extends to recombinant proteins that do not have disulfide bonds. For recombinant proteins without disulfide bonds, recombinant protein folding in the ER will consume folding resources, thus slowing down folding rates. Although the recombinant protein has no disulfide bonds, many native proteins still require disulfide bonds. Because of this, the folding to disulfide bond formation ratio will be disturbed, resulting in similar ROS stress.
To maintain an optimal ratio, either protein folding rates must increase or oxidation rates decrease. Overexpression of chaperones that increase folding capacity has successfully been used to increase protein secretion [6, 48]. For particularly large or difficult to fold proteins this may not be adequate. A new approach may be to limit the oxidation rate of Ero1p to slow down the first step of the futile cycle. This would be done in concert with repressing ERAD, as proteins would have long retention times in the ER. In this scenario, recombinant proteins would be slowly folded, albeit without high cellular stresses. This would result in longer overall process times, but may be required for difficult to fold proteins.