Functional Characterization of Solanum tuberosum ER Lumen Binding Protein (StBiP) Genes Through Complementation in Yeast kar2 Deletion Mutants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript ijms-4164889 describes complementation of loss of Kar2 (BiP) in yeast Saccharomyces cerevisiae by three isoforms of Solanum tuberosum BiP provided with yeast translocation signal. BiP is a key molecular chaperone of endoplasmic reticulum. In yeast, it is encoded by a single gene, KAR2. Its deletion is lethal. The results obtained in this work show that StBiPs can rescue the growth of yeast lacking Kar2. This is an interesting and important result. However, some experiments presented in the manuscript were performed incorrectly. Some of those experiments appear not to be crucial for the main idea of the manuscript and can be removed. However, in its present state, the manuscript cannot be recommended for publication.

 

1. Line 137. Genes cannot complement yeast. Solanum tuberosum BiP genes partially complement the loss of Kar2 in yeast or rescue kar2-Δ lethality.
2. Some designations used in the Figure 2 are misleading. It was not easy to understand that designations of the lanes refer to alleles and genetic constructs that were identified by PCR. The primers used for PCR are not mentioned in the legend. The designations of alleles need to be corrected in accordance with standard yeast genetic nomenclature (https://www.sciencedirect.com/science/chapter/bookseries/abs/pii/S0580951708703490 or https://wiki.yeastgenome.org/images/8/8f/Cherry_1995_PMID_7660459.pdf). For example, according to the standard nomenclature the designation KAR2::URA3 can be understood as replacement of KAR2 with URA3. However, these are plasmid genes that should be indicated as [KAR2 URA3]. The correct designation of KAR2 deletion allele is kar2-Δ or kar2Δ, or kar2Δ::kanMX in the case of replacement with the kanMX gene.
3. Lines 147,148. Please indicate the promoter used to drive the recombinant genes.

Lines 164-168. The description of these experiments needs to be much more detailed.

4. Line 169. Were these colonies the subclones that had lost the KAR2 plasmid?
5. Line 172. The supplementary file contains only tables. Figure S1 is not included.
6. Figure 3c. There are two diagrams with “Relative levels (ΔΔCt)” axis. What is the difference between them? Possibly, the larger one represents the levels normalized to the chromosomal KAR2 and “Kar2” represent expression of the plasmid-borne KAR2 in the kar2-Δ strain. If this is correct, why the plasmid KAR2 is expressed at the same level as the chromosomal allele? Perhaps the term “endogenous” was used to designate the plasmid-borne KAR2? In this case normalization to chromosomal wild-type KAR2 would be much more informative. This needs to be clarified. Why is there no bar for StBiP1 in the larger diagram and KAR2 in the smaller one?
7. Line 190. The heading is misleading. The statement at line 191 is wrong. Kar2 is known to protect yeast from death, since its absence is lethal. Thus, the next sentence is also invalid.
8. The panel d in Figure 4 would make sense if it possessed a negative control (e.g. a heat shock sensitive kar2 mutant). Otherwise it should be removed (as well as the section describing it). In addition, it lacks another control - the same plate but incubated at 30°C. The same applies to the panels b, c, and f.
9. Line 233. This heading is also misleading.
10. Lines 238-240. The primary mechanism of TM toxicity is inhibition of N-glycosylation. The references do not correspond to this statement.
11. The data presented in Figure 5a and b do not support the conclusions drawn. To find whether the recombinant genes confer the same resistance to TM or DTT as the endogenous Kar2, the minimal inhibitory concentrations of these agents should be determined for the kar2-Δ mutant bearing a plasmid with either one of the recombinant genes, or KAR2, as well as for the strain with the chromosomal wild-type KAR2 allele.
12. Line 271. This heading is also misleading. This section shows that the recombinant BiP do not fully complement the loss of Kar2. This leads to an increased sensitivity to oxidative stress.
13. Line 298. This heading is also misleading. The role of the Kar2 Cys63 amino acid residue has been studied previously (https://doi.org/10.1016/j.jmb.2016.08.011). The inclusion of this section is unjustified.
14. Lines 338-355. The conclusions made in this section appear to be unjustified. These experiments show that extra copies of StBiP3 provide additional protection against TM and hydrogen peroxide, whereas StBiP3 variants with Cys63 substitutions are less functional.
15. The Discussion should be much more concise and focused mainly on the results obtained.
16. I would suggest that StBiP3 does not interact with yeast Ire1. This should lead to permanent Hac1 induction. Maybe this is why HAC1 transcript does not properly react to stresses in the strain expressing this protein.

 

Typos: 

line 69 – ‘microorganisms’ – Bacteria do not have BiP

line 246 – wrong reference format

The character ß is not β

Font size in the alignment in the Figure 1 is too small. It could be at least two-fold increased to be readable.

 

Author Response

Comment 1: Line 137. Genes cannot complement yeast. Solanum tuberosumBiP genes partially complement the loss of Kar2 in yeast or rescue kar2-Δ lethality. 

Response:  We appreciate the comment pointing to careless language use on our part and changed this and other headings for accuracy.   2.2 Three Solanum tuberosum BiP genes partially complement the loss of KAR2 in yeast

Comment 2:  Some designations used in Figure 2 are misleading. It was not easy to understand that designations of the lanes refer to alleles and genetic constructs that were identified by PCR. The primers used for PCR are not mentioned in the legend. The designations of alleles need to be corrected in accordance with standard yeast genetic nomenclature (https://www.sciencedirect.com/science/chapter/bookseries/abs/pii/S0580951708703490 or https://wiki.yeastgenome.org/images/8/8f/Cherry_1995_PMID_7660459.pdf). For example, according to the standard nomenclature the designation KAR2::URA3 can be understood as the replacement of KAR2 with URA3. However, these are plasmid genes that should be indicated as [KAR2 URA3]. The correct designation of the KAR2 deletion allele is kar2-Δ or kar2Δ, or kar2Δ::kanMXin the case of replacement with the kanMX

Response:  We updated the nomenclature and appreciate the assistance in presenting the correct guidelines for designation. I have updated Figure 2 as suggested and I hope this is now correct.  I also updated the figure legend to be more informative.  I also updated the M&M adding details of screening methods. 

 Comment 3: Lines 147,148. Please indicate the promoter used to drive the recombinant genes. Kar2 and this is listed and we updated the paragraph to better describe the construction. Plese see lines 159-173:

The S. cerevisiae KAR2 is an essential gene, as haploid cells lacking a functional KAR2 are inviable [2,38]. To evaluate whether potato BiPs can substitute for Kar2p, we constructed plasmids in which StBiP1, StBiP2, and StBiP3 coding sequences were placed under the control of the KAR2 promoter, and their endogenous N-terminal signal peptides were replaced with the Kar2p signal sequence to ensure proper targeting in yeast. A point mutation was introduced into StBiP3 to convert its C-terminal YDEL to HDEL (Tyr →His) to match the canonical yeast ER-retention signal and promote efficient ER localization [1,4,39,40].

Comment 4:  Lines 164-168. The description of these experiments needs to be much more detailed. Line 169. Were these colonies the subclones that had lost the KAR2 plasmid? Line 172.  The supplementary file contains only tables. Figure S1 is not included.

Response:  We deeply considered all these comments, along with the above comments, and did extensive rewriting to better inform the reader and explain the significance of this work.  Please see lines 159-173.  Specifically, we added to the results: 

Two haploid kar2Δ::KanMX yeast strains, DGY738 and DGY740, each containing a covering URA3-marked plasmid expressing the wild-type KAR2 from the KAR2 promoter, were used for complementation testing. Plasmid shuffling was performed by transforming each strain with HIS3-marked plasmids expressing StBP1, StBIP2, and StBiP3, followed by 5-FOA counter selection to eliminate the KAR2-URA3 ‘cover’ plasmid as detailed in the Methods. This yielded six independent transformant lines. An unmodified HIS3-marked vector, referred to as the ‘empty’ plasmid, served as a plasmid-shuffling control (Figure 2a, b). For each strain-plasmid combination, ten 5-FOA-resistant colonies were screened by PCR, and a 1194 bp product confirming insertion of KanMX at the endogenous KAR2 locus. PCRs were performed immediately after transformation to identify colonies carrying both plasmids (KAR2 URA3- and HIS3- marked plasmids) and again after 5-FOA selection to confirm the loss of the URA3-marked plasmid and retention of the HIS3-marked plasmids containing KAR2, StBiP2, StBiP3, or StBiP3.  PCR products indicating the KAR2-, StBiP1-, StBiP2-, and StBiP3- containing HIS3 marked plasmid were 1400, 1900, 1500, and 2100 bp in size, respectively (Figure 2b, c, d, and Table S1). Three to four verified clones per combination were selected for further analysis. 

In M&M, we added lines 565-581.  

 Two haploid kar2Δ::KanMX yeast strains, DGY738 and DGY740, each containing a covering URA3-marked plasmid expressing the wild-type KAR2 from the KAR2 promoter, were used for complementation testing. Plasmid shuffling was performed by transforming each strain with HIS3-marked plasmids expressing StBP1, StBIP2, and StBiP3, followed by 5-FOA counter selection to eliminate the KAR2-URA3 ‘cover’ plasmid. For each strain-plasmid combination, ten 5-FOA-resistant colonies were screened by PCR using a KanMX forward primer and a 3’ KAR2 UTR reverse primer, which generated a 1194 bp product confirming insertion at the KAR2 locus. Stable presence of the KAR2-URA3 plasmid was confirmed by PCR using CovF and CovR primers, which generated a 1400 bp product ( Table S1). Stable presence of each KAR2-, StBiP1-, StBiP2-, and StBiP3- containing HIS3 marked plasmid was confirmed using M13F together with KAR2, StBiP1R, StBiP2R, or StBiP3R primers, which generated 1400, 1900, 1500, and 2100 bp products, respectively (Table S1). PCRs were performed immediately after transformation to identify colonies carrying both plasmids (KAR2-URA3 and HIS3- marked plasmids) and again after 5-FOA selection to confirm the loss of the URA3-marked plasmid and retention of the HIS3-marked plasmids containing KAR2, StBiP1, StBiP2, or StBiP3. At each step of transformation and selection, deletion of the endogenous KAR2 was also confirmed. Three to four verified clones per combination were selected for further analysis.

Finally, primers were added to Table S1. 

 Figure legend 2 was also updated:  

Figure 2 legend is lines 177-185:  Plasmid shuffle strategy and PCR verification of strain composition. (a) A schematic outlining the plasmid shuffle strategy using 5-FOA counter-selection to remove the KAR2-URA3 cover plasmid before complementation testing. (b, c, d) PCR verification of strain composition before and after 5-FOA counterselection. (b) PCR verification of the kar2::KANMX chromosomal deletion (1194 bp product) and plasmid-borne KAR2 (1400 bp product). The gel on the left shows parental strains maintained on non-selective media before transformation. The gel on the right shows PCR products from colonies immediately after transformation, which carry both KAR2-HIS3 and KAR2-URA3 plasmids, and colonies after 5-FOA selection, which retain only the HIS3-marked plasmid. (c, d) PCR verification of HIS-marked plasmids before and after selection, confirming the presence of StBiP constructs and loss of KAR2-URA3 cover plasmid. Gel images show 1900 bp, 1500 bp, or 2100 bp PCR products corresponding to StBiP1, StBIP2, or StBiP3. PCR verification of Kar2 deletion.

Comment 5:   Figure 3c. There are two diagrams with the  “Relative levels (ΔΔCt)” axis. What is the difference between them? Possibly, the larger one represents the levels normalized to the chromosomal KAR2, and “Kar2” represents expression of the plasmid-borne KAR2 in the kar2-Δ  If this is correct, why the plasmid KAR2 is expressed at the same level as the chromosomal allele? Perhaps the term “endogenous” was used to designate the plasmid-borne KAR2? In this case normalization to chromosomal wild-type KAR2 would be much more informative. This needs to be clarified. Why is there no bar for StBiP1 in the larger diagram and KAR2 in the smaller one?

Response:  

We apologize for the less-than-satisfactory description of the results.  We updated lines 204-218 and hope this is better and meets reveiwers requirements:   To assess whether expression differences contributed to these phenotypes, the KAR2 and StBiP transcript levels following 5-FOA selection were measured by qRT-PCR. We assessed KAR2 and StBiP transcript levels after 5‑FOA selection and calculated StBiP abundance relative to plasmid‑borne KAR2. Unexpectedly, StBiP1, StBiP2, and StBiP3 transcript levels were lower than KAR2, even though all genes were expressed from the same promoter and plasmid backbone (Figure 3c). StBiP2 and StBiP3 reached only approximately 35–45% of KAR2 levels, whereas StBiP1 transcripts were barely detectable. These results indicate that steady-state mRNA levels are not determined solely by the promoter context. To better compare StBiP1, StBiP2, and StBip3 transcript abundance to each other, we normalized expression to StBiP1; in this analysis (Figure 3c, insert; P <0.05), StBiP2 was approximately 6‑fold higher and StBiP3 was approximately 3‑fold higher than StBiP1.  To further validate expression of StBiP1, immunoblot analysis using a commercial antibody that can detect Kar2 and potato BiPs was performed. Blots consistently showed comparable protein levels for Kar2p, StBiP1, StBiP2, and StBiP3, despite the differences in transcript levels (Figure 3d) [38].

Comment 6:  Line 190. The heading is misleading. The statement at line 191 is wrong. Kar2 is known to protect yeast from death, since its absence is lethal. Thus, the next sentence is also invalid. The panel d in Figure 4 would make sense if it possessed a negative control (e.g., a heat shock sensitive kar2 mutant). Otherwise, it should be removed (as well as the section describing it). In addition, it lacks another control - the same plate but incubated at 30°C. The same applies to the panels b, c, and f.

Response:  The controls are present in the figures; perhaps the reviewer missed them.  But we recognized a need for a better explanation of the work.  

This is now lines 220, and the accompanying paragraph is changed for a better explanation.  Plese see lines 220 to 235: 

2.3 Kar and StBiPs transformants respond to prolonged and acute heat stress. Following 5-FOA selection and growth at 30 ˚C (Figure 4a), cells were subjected to two separate heat stress regimes: a) prolonged growth at moderately elevated temperatures, and b) acute, high-temperature heat shock. For the prolonged heat stress assay,  cells were serially diluted to YPD agar plates and incubated at 37˚C for 2 days (Figure 4b). Cells expressing wild-type Kar2p exhibited robust growth at 37˚C [41,42], whereas cells expressing StBiP1, StBiP2, or StBiP3 showed reduced growth under these conditions (Figure 4b). Immunoblot analysis of biological replicates grown at 37°C demonstrated comparable protein levels of Kar2p, StBiP1, StBiP2, and StBiP3 (Figure 4c). Together, these data indicate that potato BiPs only partially complement the heat-stress growth function of Kar2p despite being expressed at similar steady-state protein levels.  In a second acute thermosensitivity assay, exponentially growing cell cultures were exposed to 50˚C for 30 min and then spotted as serial dilutions onto YPD medium. For each strain, post-shock growth closely resembled the unstressed control, suggesting that acute 50˚C treatment did not further differentiate the growth of Kar2, StBiP1, StBiP2, or StBiP3 expressing cells (Figure 4d).

The reviewer is misunderstanding the experiment, and so we recognize a need to better explain the work in the results section.  We contrasted 2 heat treatments: one is a long sustained growth at high heat, and the other is an extremely high heat shock.  For prolonged heat stress, there were significant differences in growth despite having the same levels of protein expressing indicating that the potato BIPs did not afford the same level of heat stress protection as KAR2. For acute stress, the potato BiPs did offer the same level of protection if we compare the unstressed to stressed cells, which were presented in the same spotting assay in panel D.  The data in panel d, e, and f are all comparisons of unstressed (30 °C) and heat-stressed (50 °C).  The reviewer clearly misunderstood the experiment, so we rewrote to make it clearer. 

  Comment 7:  Line 233. This heading is also misleading.

Response:  Changed to : 2.4 KAR2 and StBiP transformants respond to chemically induced ER stress

Comment 8: Lines 238-240. The primary mechanism of TM toxicity is inhibition of N-glycosylation. The references do not correspond to this statement.

Response:  We checked and updated all references as required.  Lines 238-240.  In the section looking at BiP/HAC1 and heat stress, which I show line 247, we added references Kimata et al., 2003, Oikawa et al., 2009.  These are older but appropriate.   Regarding the Results section 2.4, we reviewed all references and updated them.  Lines 274 and 277 have new references that are more relevant and classically associated with this topic.

Comment 8:  The data presented in Figures 5a and b do not support the conclusions drawn. To find whether the recombinant genes confer the same resistance to TM or DTT as the endogenous Kar2, the minimal inhibitory concentrations of these agents should be determined for the kar2-Δ mutant bearing a plasmid with either one of the recombinant genes, or KAR2, as well as for the strain with the chromosomal wild-type KAR2.

Response:  We appreciate the comments and responded by reshaping the hypothesis and conclusions more carefully.  These are highlighted in red. 

 Comment 9:   Line 271. This heading is also misleading. This section shows that the recombinant BiP do not fully complement the loss of Kar2. This leads to an increased sensitivity to oxidative stress.

Response:   This is now  changed to 2.5 Growth of KAR2 and StBiP cells following oxidative stress

Comment 10:  Line 298. This heading is also misleading. The role of the Kar2 Cys63 amino acid residue has been studied previously (https://doi.org/10.1016/j.jmb.2016.08.011). The inclusion of this section is unjustified. The authors are correct. We were not trying to determine if KAR2 is a redox sensor, this is already known to be true. We wanted to show that yeast provides us the opportunity to evaluate whether StBIPs also perform as redox sensors.  There has been limited studies studies in plants of redox sensor for ER and in fact this work is extremely difficult to perform in this host.  The goal was to establish that we can perform thee evaluations in yeast, and open the door to new avenues of research that can tremendously benefit plant biology.  Based onthese results BiP3, which is not the best steward of HAC1 splicing, can be modified to enhance or restrict HAC1 splicing during oxidative stress.  This is extremely important, and we also show the nature of the mutations show results opposite of KAR2.   Lines 338-355. The conclusions made in this section appear to be unjustified. These experiments show that extra copies of StBiP3 provide additional protection against TM and hydrogen peroxide, whereas StBiP3 variants with Cys63 substitutions are less functional.

Response:  The strength of these concerns caused us to work on better revision and explanation of our work in the results section and introduction.  Please see the last paragraphs of the introduction to explain the point of this study.  We recognized that we needed to more directly state the goal, the limitations, and the outcomes.   

I think the point of the experiments were lost here.  The goal was to see if we can use the depth of knowledge surrounding KAR2 and extend this to investigate the functions of plant BiPs.  Given the expansion of the gene family in plants, it would see remarkable that the redundancy is complete, and that there are not functional differences among the StBIP1,2, and 3.  The goal in this study is to use KAR2 as a control to see how similar or different the plant BiPs are functionally.  This is foundational work on which we can build deeper knowledge for plant improvement.  The potential of this system can inform gene editing technologies in the future. 

We rewrote the explanation here and in the discussion.  Please see lines 271 to 293: 

In budding yeast and plants, dithiothreitol (DTT) and tunicamycin (TM ) are commonly used to induce ER stress and activate UPR responses. At low concentrations (e.g. 2 mM) DTT can robustly activate UPR whereas higher concentrations of 10 mM can cause high or acute stress accompanied by loss of viability. For TM, low doses of 0.5 µg/ml can initiate Kar-dependent ER stress protection, while higher does of 2.5 µg/ml can cause robust UPR responses accompanied by KAR2 and HAC1 induction [3,46–50]. In yeast and plants, TM-induced ER stress is accompanied by oxidative stress through increased lipid peroxidation which also impairs cell growth and viability [51–54].  In this study, exponentially growing cells were exposed to 10 mM DTT or 5µg/ml TM for one hour, serially diluted, and spotted onto YPD medium (Figure 5a, b). As expected, the overall growth for the engineered KAR2 and StBiP1, StBiP2, and StBiP3 expressing cells were comparable between chemically treated and untreated controls (Figure 5a, b). Research has already shown that KAR2 is expressed at basal “housekeeping” levels under nonstressed conditions and becomes strongly induced when TM triggers UPR.  The KAR2 promoter contains a motif known as the unfolded protein response element (UPRE) that is recognized by HAC1, a key transcriptional activator of UPR target genes, including KAR2. Because all plasmids used the same KAR2 promoter, qRT-PCR was performed to assess KAR2/BIP induction, revealing that the relative levels of KAR2, StBiP1, and StBiP2 mRNAs were consistently elevated by approximately 2.5-fold compared to untreated controls, whereas StBiP3 showed a higher induction of 4.5-fold (Figure 5c). Perhaps the cells expressing StBiP3 are experiencing higher ER stress under TM treatment than the other strains, causing UPR to be more strongly upregulated.

Then section 2.5 was rewritten to better address this commentary as well.   Please see the script.  

Comment 11: The Discussion should be much more concise and focused mainly on the results obtained. I would suggest that StBiP3 does not interact with yeast Ire1. This should lead to permanent Hac1 induction. Maybe this is why HAC1 transcript does not properly react to stresses in the strain expressing this protein.

Response:  We eliminated redundancies and refocused the discussion.  Para 1 of discussion explains the focus, rationale, and significance of this work:  Because plants have higher genetic redundancy and expanded BiP families, single gene deletions do not always lead to clear phenotypes. This can make it difficult to assign specific molecular functions to individual BiP paralogs. By expressing plant BiPs in yeast, we were able to bypass redundancy and directly test how StBiPs individually contribute to ER stress responses. Under normal conditions, the three StBiP proteins partially supported growth and colony formation of a kar2-deficient yeast strain, demonstrating that StBiPs can carry out general housekeeping functions of Kar2p. This experimental system will enable future investigations into how individual protein domains or amino acids contribute to ER chaperone activity and stress responses.

We eliminated most all discussions of IRE1 since this is a digression.   We moved the qPCR analysis up to paragraph 3 and followed this by structural modeling.   We focused only on the conclusions of this study and the value of the work.  Future work will look at IRE1 BiP interactions.   The conclusion is tightened up.

 

Comment:  Fix typo and resize Figure 1:   

Respone:  Done.  

 

Finally:  All revisions will be uploaded shortly to review the entire script

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript by Adhikari et al. presents a thorough and well-executed study utilizing a yeast heterologous complementation system to dissect the functional properties of three potato BiP paralogs (StBiP1, StBiP2, and StBiP3). The authors demonstrate that all three StBiPs can partially complement the essential yeast KAR2 gene. Through a series of stress tests (heat, ER stress induced by DTT/TM, and oxidative stress), they reveal distinct functional capacities among the paralogs, with StBiP3 showing a unique, and sometimes less efficient, role in regulating the UPR. A key finding is the functional characterization of a conserved cysteine residue (Cys63) in the ATPase domain, demonstrating its importance for oxidative stress protection in both yeast and, through transient expression, in Nicotiana benthamiana leaves. The work is significant as it provides direct experimental evidence for functional divergence among plant BiP family members, a task difficult to achieve in plants themselves due to genetic redundancy. The experimental design is sound, the data is generally clear, and the conclusions are well-supported. However, several points require clarification and revision to strengthen the manuscript before it can be considered for publication.

 

Major Comments

1. The manuscript consistently shows that StBiP3 is less effective at suppressing IRE1 endonuclease activity under non-stress conditions (higher basal HAC1 splicing). While the authors speculate on this, a more direct discussion or experimental suggestion is needed. Is this difference likely due to the unique C-terminal YDEL retention signal, subtle variations in the IRE1-interacting surface of the NBD, or an altered allosteric communication between domains?
2.  In Figure 3c and 3d, a notable discrepancy exists where StBiP1 mRNA levels are significantly lower than the others, yet protein levels appear comparable under non-stress conditions. This is an important observation that warrants a clearer explanation in the text. The authors briefly mention "differences in mRNA stability" or "feedback control of transcription" in the Discussion. They should expand on this, perhaps discussing potential differences in translational efficiency or protein stability between the StBiP proteins that could compensate for lower transcript abundance.
3. The manuscript relies heavily on endpoint RT-PCR and densitometry to calculate ratios of HAC1u to HAC1s. While informative, this method can be confounded by differences in total HAC1 transcription. A change in the ratio could reflect increased splicing or increased transcription of the unspliced form. The conclusions regarding IRE1 activity, particularly for StBiP3 under heat stress where an "opposite effect" is noted, would be strengthened by quantifying total HAC1 transcript levels (e.g., by qRT-PCR using a primer set that detects both spliced and unspliced forms) to normalize the splicing data. This would confirm whether the observed band intensities truly reflect changes in IRE1 activity or simply changes in overall transcription.

Minor Comments

1. The manuscript states that statistical analysis was performed (e.g., for qPCR data in Figures 3c, 4e, 5c, and viability in Figure 6b), but the specific statistical tests used and how significance was determined (e.g., p-values, multiple comparison corrections) are not consistently reported in the figure legends or the Materials and Methods section. This information must be added for transparency and reproducibility.
2. The text describing the HAC1 splicing results (e.g., lines 225-235, 258-268) is dense and somewhat difficult to follow. Simplifying these sentences and clearly stating the ratio changes and their implications for each strain under each condition would improve readability. For example, "In untreated cells, the ratio of HAC1u:HAC1s was 12.4 for Kar2p, indicating strong IRE1 repression. This ratio was lower for StBiP1 (3.0) and StBiP2 (2.3), suggesting a weaker interaction with IRE1."
3. Line 157-158: The sentence "Modification of a conserved cysteine... although the cell protective effects in yeast were not mimicked in plant tissues" seems to contradict the data in Figure 8, where the wild-type StBiP3 does protect plant tissues. Please rephrase for accuracy.

4. In figures, gene symbols indicating expression levels should be italicized. Please ensure this is carefully checked.

Author Response

Comment:   The manuscript consistently shows that StBiP3 is less effective at suppressing IRE1 endonuclease activity under non-stress conditions (higher basal HAC1 splicing). While the authors speculate on this, a more direct discussion or experimental suggestion is needed. Is this difference likely due to the unique C-terminal YDEL retention signal, subtle variations in the IRE1-interacting surface of the NBD, or an altered allosteric communication between domains?

Response:  In responding to this point and another made by reviewer 1, we recongize was that this paper does not directly show interaction with IRE1 and so we can only speculate here.  Clearly this is important for another study.  However, thinking about your overall comments, we wante to refocus the reader to the topic of "Subfunctionalization" of the gene family.  The questions you raise are intriguing to us and point to the value of working in yeast in order to better dissect these questions.  . To more directly address these questions we revised the abstract: 

This study evaluates the functional capacity of three potato StBiP isoforms (StBiP1, StBiP2, StBip3) to complement the kar2 deletion (kar2Δ) strain under a range of environmental and ER stress conditions. All three StBiP genes partially restored colony growth under normal conditions, demonstrating they are functional orthologs of yeast Kar2 and can support core ER housekeeping functions. Under severe stress, however, the isoforms diverged: StBiP3 most effectively complemented the kar2Δ strain during heat and chemically induced ER stress, whereas StBIp1 and StBiP2 provided weaker protection. Unfolded protein response (UPR) activation, monitored via HAC1 mRNA splicing, further highlighted isoform-specific differences in how the StBiPs support IRE1-HAC1 signaling under ER stress and oxidative stress. A conserved cysteine in the nucleotide-binding domain, previously implicated in Kar2 redox control, was also critical for StBiP3-mediated protection in yeast, although the same mutation had different consequences in plant tissues. Together these findings provide evidence for sub-functionalization among potato BiP isoforms, with StBiP3 emerging as a stress-specialized chaperone that is a promising target for improving ER stress resilience in solanaceous crops.

Next we updated the last 3 paragraphs of the introduction.  Please see lines 75-81:  To date, most evidence for subfunctionalization of plant BiPs has come from analysis of cis-acting elements in BiP promoters and the associated binding of development or stress-responsive transcription factors, which link specific environmental or developmental cues to different BiP gene expressions [20,25,31]. By contrast there is relatively little information on whether subfunctionalization also arises from structural or amino acid differences among BiP proteins themselves, raising the question of how functional diversification reflects regulatory versus protein-level divergence. 

Then lines 93-99: 

Here we use yeast as a heterologous system to functionally compare three potato BiP isoforms, StBiP1, StBip2, and StBip3. This study examines whether each isoform can replace yeast Kar2 and how well they can sustain growth and UPR signaling under heat, chemical, and oxidative ER stress. We test core chaperoning activities and stress protection potential to determine whether the potato BiP family behaves as a set of largely redundant ER chaperones or whether individual isoforms have specialized roles in maintaining proteostasis during severe stress.   

In the discussion we updated paragraph 1:  Because plants have higher genetic redundancy and expanded BiP families, single gene deletions do not always lead to clear phenotypes. This can make it difficult to assign specific molecular functions to individual BiP paralogs. By expressing plant BiPs in yeast, we were able to bypass redundancy and directly test how StBiPs individually contribute to ER stress responses. Under normal conditions, the three StBiP proteins partially supported growth and colony formation of a kar2-deficient yeast strain, demonstrating that StBiPs can carry out general housekeeping functions of Kar2p. This experimental system will enable future investigations into how individual protein domains or amino acids contribute to ER chaperone activity and stress responses.

We also updated the conclusions:  

In conclusion, this study demonstrated that yeast is an effective model to dissect the multiple biological roles of plant BiPs in relationship to similar roles of Kar2. We present evidence that the three StBiP proteins can substitute for essential Kar2 functions, while differing in their ability to manage acute and chronic heat stress, chemical ER stress, and oxidative stress. Building on extensive molecular analysis of Kar2, our data point to conserved post-translational control of BiP/kar2 at Cys63 as a determinant of ER redox protection and UPR tuning. This study also revealed isoform specific behavior in StBiP3 that is not apparent from sequence conservation alone. Considering that plants encode three or more BiPs it is worth clarifying whether expansion of this gene family in plants favors competition between BIP chaperone activities and UPR activation, like Kar2, or whether another regulatory model exists. Future work using the yeast system and complementary plant assays should clarify how individual StBiPs integrate chaperone activity, redox sensing, and UPR signaling for environmental and ER stress resilience.

 

We hope this better addresses your concerns.

Comment 2:   In Figure 3c and 3d, a notable discrepancy exists where StBiP1 mRNA levels are significantly lower than the others, yet protein levels appear comparable under non-stress conditions. This is an important observation that warrants a clearer explanation in the text. The authors briefly mention "differences in mRNA stability" or "feedback control of transcription" in the Discussion. They should expand on this, perhaps discussing potential differences in translational efficiency or protein stability between the StBiP proteins that could compensate for lower transcript abundance.

 Response:   We rewrote the explanation.  Plese see lines 203-217:  

Response:  Yes, there is much to speculate about and we have the opportunity in the follow up study to investigate these things.   We added statements to the discussion, lines 451-471 to fully point out these speculations will benefit from future work.  Here:  Regarding the endpoint RT-PCR and densitometry which provided a semi-quantitative readout of HAC1 splicing and changes in the HAC1u:HAC1s ratio, the changes in principle arise from alterations in total HAC1 transcription as well as IRE1 endonuclease activity. In this study we interpreted the shifts in HAC1u:HAC1s ratio in the context of IRE1 activity as all strains were analyzed side-by-side under identical conditions, and the direction of change was consistent across multiple treatments and biological replicates. Nevertheless, the current study does not distinguish between altered splicing efficiency and potential changes in overall HAC1 transcription.  In depth work in the future will provide insight into whether StBIPs under various stresses influence IRE1 endonuclease activity, changes in HAC1 transcription, or a combination of both

Moreover,, we used HAC1u/HAC1s mRNA ratios as a proxy for IRE1 activity and UPR signaling throughout this study. While HAC1 splicing is a necessary and conserved feature of the UPR, it does not directly measure the efficiency of StBiP1, StBiP2, or StBiP3 interactions with IRE1. In fact, very little is known in plants about how different BiPs physically interact with the IRE1 lumen domain in vivo, or whether plant IRE1 isoforms (e.g. Arabidopsis IRE1a, IRE1b, and IRE1c) can functionally substitute for yeast IRE1. Our data is consistent with individual BiPs suppressing IRE1 endonuclease activity to different degrees, but they do not directly establish the underlying binding or kinetic mechanisms.   To address this, future work will require direct testing of BiP-IRE1 interactions, cross-species complementation of IRE1 in yeast, and quantitative HAC1 as well as plant bZIP60 splicing assays.

 Comment 3:  The manuscript relies heavily on endpoint RT-PCR and densitometry to calculate ratios of HAC1u to HAC1s. While informative, this method can be confounded by differences in total HAC1 transcription. A change in the ratio could reflect increased splicing or increased transcription of the unspliced form. The conclusions regarding IRE1 activity, particularly for StBiP3 under heat stress where an "opposite effect" is noted, would be strengthened by quantifying total HAC1 transcript levels (e.g., by qRT-PCR using a primer set that detects both spliced and unspliced forms) to normalize the splicing data. This would confirm whether the observed band intensities truly reflect changes in IRE1 activity or simply changes in overall transcription.

Response:  Yes, there is much to speculate about and we have the opportunity in the follow up study to investigate these things.   We added statements to the discussion, lines 451-471 to fully point out these speculations will benefit from future work.  Here:  Regarding the endpoint RT-PCR and densitometry which provided a semi-quantitative readout of HAC1 splicing and changes in the HAC1u:HAC1s ratio, the changes in principle arise from alterations in total HAC1 transcription as well as IRE1 endonuclease activity. In this study we interpreted the shifts in HAC1u:HAC1s ratio in the context of IRE1 activity as all strains were analyzed side-by-side under identical conditions, and the direction of change was consistent across multiple treatments and biological replicates. Nevertheless, the current study does not distinguish between altered splicing efficiency and potential changes in overall HAC1 transcription.  In depth work in the future will provide insight into whether StBIPs under various stresses influence IRE1 endonuclease activity, changes in HAC1 transcription, or a combination of both

Moreover,, we used HAC1u/HAC1s mRNA ratios as a proxy for IRE1 activity and UPR signaling throughout this study. While HAC1 splicing is a necessary and conserved feature of the UPR, it does not directly measure the efficiency of StBiP1, StBiP2, or StBiP3 interactions with IRE1. In fact, very little is known in plants about how different BiPs physically interact with the IRE1 lumen domain in vivo, or whether plant IRE1 isoforms (e.g. Arabidopsis IRE1a, IRE1b, and IRE1c) can functionally substitute for yeast IRE1. Our data is consistent with individual BiPs suppressing IRE1 endonuclease activity to different degrees, but they do not directly establish the underlying binding or kinetic mechanisms.   To address this, future work will require direct testing of BiP-IRE1 interactions, cross species complementation of IRE1 in yeast, and quantitative HAC1 as well as plant bZIP60 splicing assays.

We added statements to the discussion lines 441-456: 

Regarding the endpoint RT-PCR and densitometry which provided a semi-quantitative readout of HAC1 splicing and changes in the HAC1u:HAC1s ratio, the changes in principle arise from alterations in total HAC1 transcription as well as IRE1 endonuclease activity.  In this study we interpreted the shifts in HAC1u:HAC1s ratio in the context of IRE1 activity as all strains were analyzed side-by-side under identical conditions, and the direction of change was consistent across multiple treatments and biological replicates.  Nevertheless, the current study does not distinguish between altered splicing efficiency and potential changes in overall HAC1 transcription.  To address this, future work will complement the endpoint RT-PCR assays with qRT-PCR using primer sets detecting both unspliced and spliced primers allowing normalization of ratios to the total HAC1 levels.  More in depth work will provide insight into whether StBIPs under various stresses influence IRE1 endonuclease activity, changes in HAC1 transcription, or a combination of both.  

COmment 4:  The manuscript states that statistical analysis was performed (e.g., for qPCR data in Figures 3c, 4e, 5c, and viability in Figure 6b), but the specific statistical tests used and how significance was determined (e.g., p-values, multiple comparison corrections) are not consistently reported in the figure legends or the Materials and Methods section. This information must be added for transparency and reproducibility.

Response:  We updated all figure legends and M&M to expand on the statistics used.  Simple ANOVA

Comment 5:  The text describing the HAC1 splicing results (e.g., lines 225-235, 258-268) is dense and somewhat difficult to follow. Simplifying these sentences and clearly stating the ratio changes and their implications for each strain under each condition would improve readability. For example, "In untreated cells, the ratio of HAC1u:HAC1s was 12.4 for Kar2p, indicating strong IRE1 repression. This ratio was lower for StBiP1 (3.0) and StBiP2 (2.3), suggesting a weaker interaction with IRE1."

Response: 

We rewrote the section for clarity.  These are now lines 257-271: To assess induction of UPR, we examined HAC1 mRNA splicing by endpoint RT-PCR using primers that flanked the unconventional intron, yielding a 600 nt unspliced (HAC1u) and a 348 nt spliced (HAC1s) product (Figure 4f). Band densitometry was used to obtain a HAC1u:HAC1s ratio as an indicator for the effectiveness of Kar2p and StBiPs in restricting IRE1 endonuclease activity needed to induce UPR signaling. In untreated cells, Kar2p was most effective at restricting HAC1u splicing with a ratio of approximately 12: 1. StBiP1 and StBiP2 were moderately restrictive with ratios of approximately 3:1 or 2.3 : 1, respectively, whereas StBiP3 was least restrictive and showed higher HAC1s accumulation. Following heat treatment, the KAR2 strain responded as expected with the ratio of unspliced to spliced shifting to approximately 1:1 which is consistent with robust IRE1 activation. The StBiP1 and StBiP2 strains showed only a modest shift to 2:1. or 1.4:1, indicating partial heat-induced activation of IRE1. By contrast, heat treatment of StBIP3-expressing cells increased HAC1u accumulation, suggesting that the higher HAC1 signal in this background primarily reflects increased transcription rather than enhanced splicing, and that StBIP3 is comparatively inefficient at regulating IRE1 endonuclease activity (Figure 4f).

Comment 6:  Line 157-158: The sentence "Modification of a conserved cysteine... although the cell protective effects in yeast were not mimicked in plant tissues" seems to contradict the data in Figure 8, where the wild-type StBiP3 does protect plant tissues. Please rephrase for accuracy.

Response:  Done.  

Comment 7:  Line 157-158: The sentence "Modification of a conserved cysteine... although the cell protective effects in yeast were not mimicked in plant tissues" seems to contradict the data in Figure 8, where the wild-type StBiP3 does protect plant tissues. Please rephrase for accuracy.

Response:  Gene symbols were updated throughout figures and text

Reviewer 3 Report

Comments and Suggestions for Authors

The study established that StBiP1, StBiP2, and StBiP3 from Solanum tuberosum function as orthologs of the yeast Kar2 protein. Although all three isoforms support basic cellular viability, StBiP3 proved to be the most effective chaperone, exhibiting greater resilience under severe environmental challenges, such as heat and chemically induced ER stress. The research provides strong evidence for the sub-functionalization of BiP isoforms in potatoes. StBiP3 likely plays a specialized role in maintaining protein homeostasis during acute stress, whereas StBiP1 and StBiP2 may handle more routine folding tasks. This manuscript bridges a gap in our understanding of how potato crops respond to ER stress at the molecular level. The authors have conducted solid work, and their findings are solid. By pinpointing the specific strengths of StBiP3, they have identified a promising target for future crop improvement strategies aimed at enhancing abiotic stress tolerance in solanaceous species.

Author Response

Thank you

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The text of the manuscript has been significantly improved. However, this has revealed additional problems in the manuscript. Moreover, the reviewer’s main concerns have not been addressed.

(i) The panel (b) in Figure 4 lacks a control. Panel (a) is not an appropriate control in this case. It should show the same spotting assay incubated at 30 degrees (as Figure 3B).

(ii) Growth at 37°C is a heat stress. Incubation at 50°C for 30 min is a heat shock. The presented data on heat shock sensitivity do not allow one to conclude that the sensitivity of the transformants bearing the heterologous genes is the same as in the transformant with endogenous KAR2. Spotting does not provide precise data on the survival rate. Possibly, increasing the incubation time to achieve a much higher cell death rate would yield more convincing data. Alternatively, the survival rate can be estimated by counting colony-forming units (CFU) before and after incubation.

(iii) The assessment of TM and DTT sensitivities was performed incorrectly. To do this, the minimal inhibitory concentrations of these compounds in liquid medium should be determined for each strain. Alternatively, colony growth should be assessed on solid medium containing TM or DTT at concentrations that significantly inhibit growth even of the transformant expressing KAR2.

Other points.

1. It is still unclear why the proportion between BiP1-3 ΔΔCt differs before and after normalization. Something is wrong here.
2. Different primer pairs were used for qRT-PCR for each gene. It is possible that different primers provide different PCR efficiencies. This may lead to an incorrect assessment of differences in the mRNA levels between these genes.
3. 222-223: The efficacy of Western blotting with a polyclonal antiserum against GRP78 may also differ among BiPs. Therefore, differences in band intensity may not reflect differences in protein abundance.
4. 313-317: The end point RT-PCR does not provide a quantitative assessment of the HAC1s:HAC1i ratio. This should be discussed using more precise language.
5. 330-331. BiPs do not have ‘oxidative stress functions’.
6. What is XTT?
7. 377,378. Kar2/BiP does not protect the cell from oxidative stress. Its insufficient function induces stress itself, thereby makes the cell more vulnerable to oxidative stress.
8. 387: The expression ‘non-stressed and treated conditions’ sounds weird.
9. 389-391: This conclusion contradicts the data presented.
10. 441: What is ‘high protein sequence’? Perhaps the term ‘homology’ is missing?

Conclusion:

Despite this criticisms, the reviewer still believes that the manuscript contains some interesting and important data (e.g. data on Cys63 substitutions). However, in its present state, it cannot be recommended for publication.

Author Response

We appreciate the diligence of Reviewer 1 in carefully going through our manuscript to make sure it is the highest quality that we all desire.  In fact, we submitted the article to the journal’s English language editing for script and figures and have adopted all suggestions.  Below are the comments from Reviewer 1 and our responses:

 

Comment (i): The panel (b) in Figure 4 lacks a control. Panel (a) is not an appropriate control in this case. It should show the same spotting assay incubated at 30 degrees (as Figure 3B).

 Response:  We disagree with Reviewer 1 that a 30˚C spotting plate is required to interpret the data presented in Figure 4b. The growth of all strains at 30˚C is already presented in Figure 3B. Moreover, an abbreviated assay streaking equal amounts of liquid cultures onto a plate in Figure 4a reiterates the same outcome and sets the stage for Figure 4b. This presentation is to avoid the appearance of reusing the same data in two figures (i.e. performing the same spotting assay as in Figure 3 without adding any new information). For long term growth at 37˚C, the appropriate comparative control is the strain expressing the S. cerevisiae KAR2 gene, which we included at the top of the same plate. We would also point out that we have repeated these assays at both 30˚C and 37˚C multiple times and are confident that the images presented in Figures 3 and 4 accurately represent the growth pattern of these strains. 

 

Comment (ii): Growth at 37°C is a heat stress. Incubation at 50°C for 30 min is a heat shock. The presented data on heat shock sensitivity do not allow one to conclude that the sensitivity of the transformants bearing the heterologous genes is the same as in the transformant with endogenous KAR2. Spotting does not provide precise data on the survival rate. Possibly, increasing the incubation time to achieve a much higher cell death rate would yield more convincing data. Alternatively, the survival rate can be estimated by counting colony-forming units (CFU) before and after incubation.

Response: The role of UPR is in cell survival and adaptation to stress.  The overarching goal is to determine if the plant BiP proteins afford UPR protection and this does not require pushing cells toward death, although it is clear in Figure 4b, that there are different qualities to survival.  The differences in heat sensitivity are clear and we did not overstate our conclusions here.  A higher cell death rate by pushing the system more would not allow us to see the qualitative differences among the plant proteins which is our goal.

We agree with Reviewer 1 that the data in Figure 4d does not ‘allow one to conclude that the sensitivity of the transformants bearing the heterologous genes is the same as in the transformant with the endogenous KAR2. As we indicate in the manuscript, short term exposure to 50˚C did not lead to any significant differences in growth compared to their unstressed control. While CFU analysis would provide a quantitative measure of the data, this does not present itself as a reasonable line of further investigation given the little difference identified in the spotting assays. We feel that it is important to point out that long term heat treatment results in significant metabolic reprogramming while short term heat shock requires adaptive changes.  These data show very well that the plant BiPs can complement heat shock, and not prolonged stress requiring metabolic reprogramming.  This suggests that BIP can support some but not all functions, perhaps partner interactions.  Future work to understand the similarities and different partner interactions would benefit from the contrasting heat stress conditions.  These are assay that we can build on very nicely.  We explain this in Discussion lines 494-501.

 

Comment (iii). The assessment of TM and DTT sensitivities was performed incorrectly. To do this, the minimal inhibitory concentrations of these compounds in liquid medium should be determined for each strain. Alternatively, colony growth should be assessed on solid medium containing TM or DTT at concentrations that significantly inhibit growth even of the transformant expressing KAR2.

Response: We disagree with the recommendation made by Review 1 that an MIC assay is the appropriate measure of cell sensitivity to TM and DTT. The purpose of these spotting assays were not to identify the concentration of these agents that kill/inhibit cellular growth, rather to show that the concentrations of these agents and the exposure times used did not impact cell viability. Therefore, any changes in KAR2/StBiP transcript levels and HAC1 processing that results from this treatment regime can be attributed to a UPR response and not cell death. The conditions (concentration and exposure times) are the same for the spotting, qPCR,  and the end-point PCR analysis which is now clearly stated in the Materials and Methods, lines 633-638.

 

Other Points

Comment 1: It is still unclear why the proportion between BiP1-3 ΔΔCt differs before and after normalization. Something is wrong here. 

Response:  We have revised Figure 3c and Figure 3d and switched panels for better logic.  In panel 3c, we present another view of the same gel with more of the upper part present where a very, very faint pre-processed Kar2 band is barely detectable across all StBiPs.  These findings support proper maturation of proteins.  In panel 3d, we changed the qRT-PCR data to introduce a value representing the relative levels of StBiP1 (0.004).  This allows us to directly state the relative measures.  We have edited the explanation in lines 218-222 to better explain the relationships directly. 

Comment 2. Different primer pairs were used for qRT-PCR for each gene. It is possible that different primers provide different PCR efficiencies. This may lead to an incorrect assessment of differences in the mRNA levels between these genes.

Response:  All primer pairs used in this study were validated using a 10-fold template dilution assay prior to qRT-PCR analysis. Standard curves were generated to determine the amplification efficiencies.  The efficiencies ranged between  90–100%. Gene expression levels were normalized to the reference gene and calculated using the ΔΔCt method, which helps minimize the potential influence of efficiency variation. Similar statement is added to the M&M lines 686-689. 

Comment 3. 222-223: The efficacy of Western blotting with a polyclonal antiserum against GRP78 may also differ among BiPs. Therefore, differences in band intensity may not reflect differences in protein abundance.

Response: The immunoblot data is now only used to confirm protein expression for each of the BiPs and we have removed the suggestion that there are no differences in overall protein expression levels between the Kar2p/StBiPs.   

Comment 4:  313-317: The end point RT-PCR does not provide a quantitative assessment of the HAC1s:HAC1i ratio. This should be discussed using more precise language.

Response: The same primer pair was used to amplify both HAC1 isoforms in a single reaction. As they sit in flanking exons, 2 bands will be detected on a gel. Clear ratio differences will represent a strong shift in splicing, and this is “semiquantitative”.  The ratio shift from 12:1 to 1:1 for KAR2 is undeniably a ratio shift. The plant proteins show less of a shift pointing out that its ability to regulate HAC1 splicing is less than KAR2.   We could try to develop isoform specific primers for a “true molar ratio” of spliced to unspliced. But for the purpose of this study, we can clearly state that StBiPs do not replace KAR2 for regulating HAC1 splicing with the same kinetics. As requested, we have updated the language that describes our analysis approach (lines 272-290).   

There are many studies reporting semiquantitative RT-PCR in plants, humans and yeast and this is done by comparing ratios to each other and internal controls PCR products (actin in this case). In fact, we have published this recently for the plant bZIP60 splicing downstream of IRE1. 

doi: 10.1251/bpo20---Semiquantitative RT‑PCR analysis to assess the expression levels of multiple transcripts from the same sample – Biol Proced Online (2001)

doi: 10.1007/978-1-0716-4901-5_20.     Semi‑quantitative RT‑PCR Assay for the Analysis of Alternative Splicing of Interleukin Genes” – Methods Mol Biol (2025).

doi: 10.1111/nph.19882   bZIP60 and Bax inhibitor 1 contribute IRE1-dependent and independent roles to potexvirus infection – New Phytologist (2024)

doi:10.1371/journal.pgen.1005164  The UPR Branch IRE1-bZIP60 in Plants Plays an Essential Role in Viral Infection and Is Complementary to the Only UPR Pathway in Yeast – PLOS Genetics (2015)

 

Comment 5:  330-331. BiPs do not have ‘oxidative stress functions’.

Response:  We have edited this statement to : “the oxidative stress protection provided by individual StBiP isoforms”

Comment 6. What is XTT?

Response:  The chemical name is 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.  We have now included a brief description of the XTT viability assay in the Materials section of the manuscript.

Comment 7, 8, and 9 : 377,378. Kar2/BiP does not protect the cell from oxidative stress. Its insufficient function induces stress itself, thereby makes the cell more vulnerable to oxidative stress.  387: The expression ‘non-stressed and treated conditions’ sounds weird. 389-391: This conclusion contradicts the data presented.

Response: We agree and conclude that the differences in H₂O₂-dependent loss of cell viability indicates that StBiPs are less efficient at protecting cells against such stress than Kar2.  We understand this comment to suggest that our explanation is not entirely clear or consistent so we rewrote the entire section, now lines 383 -410. 

 

Comment 10: 441: What is ‘high protein sequence’? Perhaps the term ‘homology’ is missing?

Response:  We have edited this sentence to include the word homology, as suggested. 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Accept

Author Response

Comment 1:  accept

Response:  Thank you.   We also completed English language editing on Friday and are including these to address the "can improve" areas of the comments

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The reviewer disagrees with the arguments presented in the authors’ responses.

 

(i) Figure 4b requires proper control. The explanation that “This presentation is to avoid the appearance of reusing the same data in two figures (i.e. performing the same spotting assay as in Figure 3 without adding any new information)” is not valid. The figures can be rearranged to combine all spotting assays in a single separate figure.

(ii) The death of a portion of cells in the control would show that the heat shock works. 

 

(iii) There is no explanation that the spotting after DTT and TM treatments was intended “to show that the concentrations of these agents and the exposure times used did not impact cell viability” as was stated in the response.

 

Other points:

1. Regarding Figure 3c and d, it is hard to believe that a 0.4% mRNA level can provide a comparable protein level according to the Western blot, which also compensates for the loss of endogenous Kar2. I am afraid that there may be an experimental error in the mRNA analysis.
2. The XTT assay provides data on overall metabolic activity in cell suspensions. Cells can be inviable (i.e., unable to grow and form a colony) and retain metabolic activity. Therefore, the designation of the axis as “Cell viability” is incorrect. The actual data on cell viability can be obtained by counting CFU, which is also very simple and does not require any special reagents or equipment.

Author Response

We thank the reviewer for their continued willingness to critically review our manuscript. We address each of their comments below.

Comment 1:  Quality of English must be improved:   
Response:  As the manuscript has undergone English language editing using the services provided by the journal, it is unclear what additional improvement is being requested by the reviewer.

Comment 2: Figure 4b requires proper control. The explanation that “This presentation is to avoid the appearance of reusing the same data in two figures (i.e. performing the same spotting assay as in Figure 3 without adding any new information)” is not valid. The figures can be rearranged to combine all spotting assays in a single separate figure.

Response 2: As requested, we have rearranged the figure to include the corresponding 30˚C plate that was  carried out in parallel with the 37˚C one.  This is now panel A. 

 

Comment 3: The death of a portion of cells in the control would show that the heat shock works.

Response 3:  We recognize that we stated loss of viability as a critical expectation of heat shock and this would be consistent with loss of BiP1 viability at 37˚C.  However, our treatment regime for 50˚C exposure resulted in little loss in viability as assayed using a spotting assay.  We attribute this to the methodology used for heat exposure. The reviewer is correct that we need to do a better job of explaining that our goal here was to focus on stress induction not loss of viability.  For someone reading the manuscript, reduced growth would be expected. We have edited the text to better reflect our aim. We have also eliminated statements concerning KAR2 performing “as expected”.   KAR2 is the control for contrasting how plant BiPs are performing and we recognize our choice of word might express our optimism rather than our certainty.

 

While we agree that data from a CFU analysis would support that the heat shock worked, we believe that the increase in KAR2 transcript levels following acute heat exposure is a molecular demonstration that a heat shock response was activated. With the reviewer’s concern over the growth pattern for 50˚C plated cells, after considering the entirety of the work in the figure and to avoid confusion, we elected to remove the spotting plates. This better draws the attention of the reader to the molecular data showing the responsiveness of the StBiP genes to acute short term heat treatment. We have also added additional details within the Methods section to better state how these experiments were carried out (lines 635-636).

 

Comment 4:  There is no explanation that the spotting after DTT and TM treatments was intended “to show that the concentrations of these agents and the exposure times used did not impact cell viability” as was stated in the response.

 

Response 4:   We had stated that exposure to high levels of (10mM) DTT or (5µg/ml) TM will lead to loss of viability, and this is an error in our explanation.  Our goal is to show that StBIPs and Kar2 contribute to stress (heat, chemical) responses in a measurable way. In some instances this is represented by spotting assays and in others this is represented by molecular studies.  We recognize now that for someone reading the manuscript, reduced growth would be expected given our original statements. We have edited the text to remove this confusion. 

 

Modified statements in lines 291-295: “Here, cells were exposed for a short period of time to DTT or TM to induce ER stress while minimizing cell death.  Serial dilution spotting onto YPD medium demonstrated that overall growth of KAR2- and StBiP1-, StBiP2-, and StBiP3-expressing cells was comparable between the chemically treated and untreated controls (Figure 5a, b).”

Other points:

Comment 1:  Regarding Figure 3c and d, it is hard to believe that a 0.4% mRNA level can provide a comparable protein level according to the Western blot, which also compensates for the loss of endogenous Kar2. I am afraid that there may be an experimental error in the mRNA analysis.

Response 1: We completely agree with the reviewer that it is unexpected to find comparable BiP protein levels in a strain that has 0.4% of the relative mRNA transcript present. While we have not determined the protein half-life for the StBiPs expressed in the yeast background, it is reported that the wildtype Kar2 protein is extremely stable with a half-life of 27 hours (https://yeastgenome.org/locus/S000003571/protein). This likely contributes to our observation and is something that we plan on testing in future work. To help the reader interpret our findings, we have added the average Cq values for KAR2 and StBiPs into the figure legend (line 215). 

Comment 2: The XTT assay provides data on overall metabolic activity in cell suspensions. Cells can be inviable (i.e., unable to grow and form a colony) and retain metabolic activity. Therefore, the designation of the axis as “Cell viability” is incorrect. The actual data on cell viability can be obtained by counting CFU, which is also very simple and does not require any special reagents or equipment.

Response 2: We agree with the reviewer’s point and have changed the y-axis label for the graph presented in Figure 6b to “XTT metabolic activity (%)”.

The following text has also been edited to reflect this change

Lines 350-353: To quantify this effect, a colorimetric XTT metabolic activity assay was carried out, revealing an approximately 10-20% reduction in metabolic activity for KAR2, StBiP1, and StBiP3 cells and an over 40% reduction in activity for StBiP2 cells.

Lines 357-358: Thus, the differences in H₂O₂-dependent loss of metabolic activity indicates that StBiPs are less efficient at protecting cells against such stress than Kar2.

Line 363: Quantitative determination of metabolic activity following H₂0₂ exposure.

Line 643: To determine the percent of metabolically activity cells in the culture, peroxide treated and control cells were pelleted and resuspension in 1 mL phosphate buffer saline (PBS).