Identification of Aly1 and Aly2 as Modulators of Cytoplasmic pH in Saccharomyces cerevisiae
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsReview for
Identification of Aly1 and Aly2 as modulators of cytoplasmic pH in yeast
By Guoyong Liu1, †, Xiuli Han1, †, Xiang Yu1, Yu Wang1, Jinbiao Ma1 and Yongqing Yang1, 3*
The regulation of intracellular pH in yeast (Saccharomyces cerevisiae) cells is critical for cell 7 function and viability. In yeast, protons (H+) can be excreted from cell by the plasma membrane 8 ATPase Pma1 and pumped into vacuoles by the vacuolar H+-ATPase. Because Pma1 is critical to the 9 survival of yeast cells, it is unknown whether other compensatory components are involved in the 10 pH homeostasis in the absence of Pma1.
Very strange that this was not investigated earlier, good idea to do it
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Add
Saccharomyces cerevisiae
In title
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After repeated screenings and verification, we identified two proteins, ALY1 14 and ALY2, that play a role in the regulation of intracellular pH when Pma1 is deficient. Our research 15 uncovers a new perspective on the regulation of intracellular pH related to Pma1 and also preliminarily reveals a role for ALY1 and ALY2 in the regulation of intracellular pH.
An alignment of 17 deduced protein sequences from plant, fungi, and ciliate H+-ATPase genes
Wach, A., Schlesser, A., Goffeau, A.
Journal of Bioenergetics and Biomembranes, 1992, 24(3), pp. 309–317
Only one ATPase in Saccharomyces cerevisiae?
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Aly1 and Aly2 belong to the α-arrestin family of proteins, which comprises 14 members in budding yeast. This family serves as trafficking adaptor proteins in the regulation of signal-induced PM protein endocytosis and intracellular sorting of nutrient permeases [24, 25]. α-arrestins are also named arrestin-related trafficking adaptors (ARTs)
Bit confusing with aly alginate lyase gene (aly)
Characterization of catabolite repression and the promoter of the alginate lyase gene (aly) from Pseudomonas sp.
Kim, G.U.-T., Kim, J.-M., Yu, J.-H., Kong, I.-S.
Biotechnology Letters, 1996, 18(11), pp. 1271–1276
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In our study, the deficient function of Pma1 resulted in a 300 lower pH in the cytosol; whether V-ATPase activity can be stimulated by the observed 301 lower intracellular pH in the absence of Pma1 function is unknown
Interesting observation, should be investigated in your next study
Author Response
Response to reviewer 1:
Question 1: Add Saccharomyces cerevisiae in title.
Response: Thank you for your suggestion. We have added Saccharomyces cerevisiae in title of the manuscript.
Question 2: After repeated screenings and verification, we identified two proteins, ALY1 and ALY2, that play a role in the regulation of intracellular pH when Pma1 is deficient. Our research uncovers a new perspective on the regulation of intracellular pH related to Pma1 and also preliminarily reveals a role for ALY1 and ALY2 in the regulation of intracellular pH.
An alignment of 17 deduced protein sequences from plant, fungi, and ciliate H+-ATPase genes,Wach, A., Schlesser, A., Goffeau, A. Journal of Bioenergetics and Biomembranes, 1992, 24(3), pp. 309–317, Only one ATPase in Saccharomyces cerevisiae?
Response: We have read the paper you recommended, and we are sorry that our manuscript is not clear enough. There are two ATPase in Saccharomyces cerevisiae, PMA1 and PMA2. According to previous reference (Carmelo, V., Bogaerts, P., Activity of plasma membrane H+-ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at optimal and low pH, Arch Microbiol, 1996, 166,5: 315-320), PMA1 is the essential plasma membrane ATPase, and PMA2 is non-essential due to its lower expression level.
Question 3: Aly1 and Aly2 belong to the α-arrestin family of proteins, which comprises 14 members in budding yeast. This family serves as trafficking adaptor proteins in the regulation of signal-induced PM protein endocytosis and intracellular sorting of nutrient permeases [24, 25]. α-arrestins are also named arrestin-related trafficking adaptors (ARTs) Bit confusing with aly alginate lyase gene (aly). Characterization of catabolite repression and the promoter of the alginate lyase gene (aly) from Pseudomonas sp. Kim, G.U.-T., Kim, J.-M., Yu, J.-H., Kong, I.-S. Biotechnology Letters, 1996, 18(11), pp. 1271–1276.
Response: Thank you for your question. It is indeed easy to confuse that some different proteins or genes have the same abbreviations, such as arrestin-related trafficking adaptors (ARTs) in this manuscript and alginate lyase gene (aly) you mentioned, and also Ser/Thr protein kinase SOS2-LIKE PROTEIN KINASE5 (PKS5) and polyketide synthases 5 (PKS5) in the reference reported.
Question 4:
In our study, the deficient function of Pma1 resulted in a lower pH in the cytosol; whether V-ATPase activity can be stimulated by the observed lower intracellular pH in the absence of Pma1 function is unknown.
Interesting observation, should be investigated in your next study.
Response: Thank you for your recognition for this study. The coordination of P-ATPase and V-ATPase to maintain cytosolic pH homeostasis is important for yeast growth. When P-ATPase is deficient, it is still unclear how V-ATPase activity is regulated. We will further explore this issue.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript by Liu and co-workers (Identification of Aly1 and Aly2 as modulators of cytoplasmic pH in yeast) is certainly within the scope of Current Issues in Molecular Biology. In general the manuscript is well written (although some parts need revision) and brings new and interesting results. Initially they screened a deletion library in fission yeast with compound PS-1, and then they moved to budding yeast to confirm one of the identified genes (no explanation is given for this change in yeast species). Moreover, some issues need to be addressed before the manuscript is considered for publication:
-L27-28 needs revision (very confusing).
-L36-38 the sentence regarding Debaryomyces hansenii and NaCl stress is out of context, it should be removed.
-L108 “the S. pombe haploid deletion mutant set ver 4.0 (M-4030H)” needs a reference describing its construction, and from where the set was purchased/obtained.
-L147 “We deleted each of these 16 genes in the RS-72 background” but they showed only the results for the ALY1/ALY2 genes….. other deletions should be shown (even if they gave “negative” results).
-Figure 2 and its legend (L156-162)…. In figures A, B, and C there is a third panel showing glucose/galactose + Hyg…. which is not explained at all. The meaning/significance of such panels needs a description/explanation.
-An important result that needs to be included in Figure 2 is how the deletion of Aly1 or Aly2 in the budding yeast strain AH109 strain influences the resistance/susceptibility to compound PS-1 (the one used in their screen).
-L170-172 the authors claim that the strains deleted on Aly1 or Aly2 in the AH109 strain background could not grow in galactose-containing medium (Figure 2C). This is strange because if strain RS-72 (galactose positive) is derived from strain AH109, this last strain (and its aly1/aly2 derivatives) should also be galactose positive! Indeed, the description of the genes/genome of the AH109 and RS-72 (L311-312) strains is very superficial (two strains, but only one genotype is shown). How the ProGAL:Pma1 construct was introduced into strain RS-72? in a plasmid? or integrated into the genome? how? which selectable marker was used to identify the correct transformants? more details are needed.
-Figure 3 needs revision. In Figures 3A and 3B the authors claim that the strains/colonies “acquired yellow halos around them, indicative of medium acidification and thus H+ extrusion”. However, this reviewer could not see any “yellow halos” in their pictures. Authors need to better show their results (maybe using color figures, not black & white pictures). The data shown in Figures 3C and 3D is also hard to see, although there are 4 alternatives (strains and carbon source) in each figure, this reviewer can only see clearly 3 curves….. authors need to use bigger symbols to clearly show the differences between strains and carbon sources.
-Figure 4A and 4B….. which strain was used in these figures? no idea! Figure 4C shows the data for strain RS-72 on glucose…. how this is possible, if strain RS-72 cannot grow on glucose? details are missing!
-Item 4.2 (Materials and Methods) needs revision (deletion assay?). Indeed, authors need to give full details (primers/plasmid/genes used) on how aly1 and aly2 deletion strains were obtained, so that other authors can reproduce their results/experimental approach (including how the deletions were confirmed).
-Finally, what is the composition/structure of compound PS-1? Where can it be obtained? it is commercially available?
-The list of references needs revision, they are not formatted (some have full journals names, others abbreviated), and some are incomplete (missing page/article numbers).
Comments on the Quality of English LanguageSee above
Author Response
Response to reviewer 2:
Question 1: -L27-28 needs revision (very confusing).
Response: We are sorry for the confusing. According to your suggestion, we have revised this sentence (line 25-29 in the revised manuscript).
Question 2: -L36-38 the sentence regarding Debaryomyces hansenii and NaCl stress is out of context, it should be removed.
Response: Thanks for your suggestion. According to your suggestion, we have removed this sentence.
Question 3:-L108 “the S. pombe haploid deletion mutant set ver 4.0 (M-4030H)” needs a reference describing its construction, and from where the set was purchased/obtained.
Response: The S. pombe haploid deletion mutant set ver 4.0 (M-4030H) was purchased from BIONEER company, and we are sorry that we did not find any reference citation related to this set.
Question 4:-L147 “We deleted each of these 16 genes in the RS-72 background” but they showed only the results for the ALY1/ALY2 genes….. other deletions should be shown (even if they gave “negative” results).
Response: Thanks for your suggestion. According to your suggestion, we have supplemented other deletions (Figure S1 in the revised manuscript).
Question 5:-Figure 2 and its legend (L156-162)…. In figures A, B, and C there is a third panel showing glucose/galactose + Hyg…. which is not explained at all. The meaning/significance of such panels needs a description/explanation.
Response: The third panels are the controls to illustrate that Aly1 or Aly2 were the knocked-out mutants, which used hygromycin as a screening marker. According to your suggestion, we have added this explanation in the figure legend of figure 2 in the revised manuscript (line 160-163 in the revised manuscript).
Question 6:-An important result that needs to be included in Figure 2 is how the deletion of Aly1 or Aly2 in the budding yeast strain AH109 strain influences the resistance/susceptibility to compound PS-1 (the one used in their screen).
Response: Thank you for your constructive suggestion, we have supplemented this result in Figure S2 in the revised manuscript. The growth of AH109, aly1Δ and aly2Δ can also be inhibited by PS-1, but aly1Δ and aly2Δ show better growth than AH109 strain.
Question 7:-L170-172 the authors claim that the strains deleted on Aly1 or Aly2 in the AH109 strain background could not grow in galactose-containing medium (Figure 2C). This is strange because if strain RS-72 (galactose positive) is derived from strain AH109, this last strain (and its aly1/aly2 derivatives) should also be galactose positive! Indeed, the description of the genes/genome of the AH109 and RS-72 (L311-312) strains is very superficial (two strains, but only one genotype is shown). How the ProGAL:Pma1 construct was introduced into strain RS-72? in a plasmid? or integrated into the genome? how? which selectable marker was used to identify the correct transformants? more details are needed.
Response: The strain RS-72 was not derived from strain AH109, it was derived from strain BWG1-7. ProGLC:Pma1 in the strain of BWG1-7 was replaced with ProGAL:Pma1 in the strain of RS-72 in the genome. The more detailed information of the construction of the strain RS-72 could be checked in the reported reference (Cid, Angel, Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences, CURR GENET, 1987, 12, 105-110).
Question 8:-Figure 3 needs revision. In Figures 3A and 3B the authors claim that the strains/colonies “acquired yellow halos around them, indicative of medium acidification and thus H+ extrusion”. However, this reviewer could not see any “yellow halos” in their pictures. Authors need to better show their results (maybe using color figures, not black & white pictures). The data shown in Figures 3C and 3D is also hard to see, although there are 4 alternatives (strains and carbon source) in each figure, this reviewer can only see clearly 3 curves….. authors need to use bigger symbols to clearly show the differences between strains and carbon sources.
Response: Thanks for your suggestions. As to Figure 3A and 3B, we show results in color figures and readjust contrast ratio to better show the results; As to Figure 3C and 3D, we also show results in color figures to show the differences between strains and carbon sources.
Question 9:-Figure 4A and 4B….. which strain was used in these figures? no idea! Figure 4C shows the data for strain RS-72 on glucose…. how this is possible, if strain RS-72 cannot grow on glucose? details are missing!
Response: The strain of AH109 was used in Figure 4A and 4B. As to the growth of RS-72 on glucose in Figure 4C, RS-72 can survive on glucose culture, but with a more slowly growth rate, which was evidenced from Figure 2C and previously reported reference (Cid, Angel, Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences, CURR GENET, 1987, 12, 105-110). According to your suggestion, we have added the detailed information of this experiment in the figure legend of figure 4 with the tracked changes in the revised manuscript.
Question 10:-Item 4.2 (Materials and Methods) needs revision (deletion assay?). Indeed, authors need to give full details (primers/plasmid/genes used) on how aly1 and aly2 deletion strains were obtained, so that other authors can reproduce their results/experimental approach (including how the deletions were confirmed).
Response: Thanks for your suggestions. We used homologous recombination method to produce aly1 and aly2 deletion strains. According to your suggestion, we have added primer information as shown in table S1, and we have added the information of approach to produce the mutants in the method part (line 348-354 in the revised manuscritp).
Question 11:-Finally, what is the composition/structure of compound PS-1? Where can it be obtained? it is commercially available?
Response: The structure of PS-1 was shown as listed below. PS-1 was synthesized and was not commercially available now. The detailed information of PS-1 could be checked in our previous published paper. (Yang, Y., Testing the polar auxin transport model with a selective plasma membrane H+-ATPase inhibitor, J Integr Plant Biol, 2022, 64, 1229-1245.)
PS-1 structure
Question 12:-The list of references needs revision, they are not formatted (some have full journals names, others abbreviated), and some are incomplete (missing page/article numbers).
Response: Thanks for your suggestions. We have revised the references with the abbreviated names, and also revised the incomplete references. The references are revised with tracked changes in the revised manuscript.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAlthough the authors have attended the majority of issues raised by this reviewer, there are still things that need to be improved:
-L231-232 needs revision (it does not describe what was done to obtain Fig. 4C).
-the source of the S. pombe haploid deletion mutant set ver 4.0 (purchased from BIONEER company?) should be clearly described in Materials and Methods (L336-337).
-L337-339 the description of the genome of strain AH109 is somehow confusing and does not follow standard gene description in S. cerevisiae. What is the meaning of “LYS2::GAL1UAS-GAL1TATA-HIS3”, “GAL2UAS-GAL2TATA-ADE2”, or “URA3::MEL1TATA-lacZ MEL1”? what is “UAS” and “TATA”? are the genes deleted? (in this case they should be in lowercase), or introduced? it is not clear!
-L352 the source/reference for plasmid pAG32 should be described in detail.
Comments on the Quality of English LanguageSee above
Author Response
Response to reviewer 2 (Round 2):
Question 1: -L231-232 needs revision (it does not describe what was done to obtain Fig. 4C).
Response: Thanks for your suggestion, we have removed this sentence in the revised manuscript.
Question 2: -the source of the S. pombe haploid deletion mutant set ver 4.0 (purchased from BIONEER company?) should be clearly described in Materials and Methods (L336-337).
Response: Thanks for your suggestion. According to your suggestion, we have described the source detail in Materials and Methods (L335-336).
Question 3:-L337-339 the description of the genome of strain AH109 is somehow confusing and does not follow standard gene description in S. cerevisiae. What is the meaning of “LYS2::GAL1UAS-GAL1TATA-HIS3”, “GAL2UAS-GAL2TATA-ADE2”, or “URA3::MEL1TATA-lacZ MEL1”? what is “UAS” and “TATA”? are the genes deleted? (in this case they should be in lowercase), or introduced? it is not clear!
Response: Thanks for your suggestion, we are sorry for the confusing. According to your suggestion, we have revised this description and supplemented the more information of AH109 yeast strain in the Materials and Methods (L341-345).
Question 4:-L352 the source/reference for plasmid pAG32 should be described in detail.
Response: Thanks for your suggestion. According to your suggestion, we have supplemented the new reference in the revised manuscript (reference [35] in the revised manuscript).
