A double role of the Gal80 N terminus in activation of transcription by Gal4p

Activation of gene expression by Gal4p in K. lactis requires an element in the N terminus of KlGal80p that mediates nuclear co-import of KlGal1p and galactokinase inhibition to support the co-inducer function of KlGal1p.

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Thank you for this interesting contribution to Life Science Alliance. We are looking forward to receiving your revised manuscript. --An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, http://www.life-sciencealliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title and running title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.

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Full guidelines are available on our Instructions for Authors page, http://www.life-sciencealliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** The authors have identified an NLS at the N-terminus of KlGal80, the inhibitor of KlGal4. The authors have fused KlGal80 to GFP and found that while wild-type Gal80 localized to the nucleus, Gal80 with the mutated NLS K5A/R6A was not localized to the nucleus but present everywhere inside the cell. One would expect that the KlGal80K5A/R6A mutant strain was deficient for inhibition of KlGal4, however, the authors have found that it is actually deficient for induction, as the presence of the mutant KlGal80K5A/R6A prevented activation of beta-galactosidase under both repressing (glucose) and inducing conditions (galactose). The authors have found that the overexpression of Gal1 restores the induction of beta-galactosidase in the KlGal80K5A/R6A mutant strain and the authors have shown that the K5A/R6A mutation reduces the interaction of KlGal80 with KlGal1. The authors conclude that the NLS serves two functions: it localizes KlGal80 to the nucleus, where it binds and inhibits KlGal4, and it interacts with KlGal1, which serves to switch the KlGal80-KlGal4 complex to the KlGal80-KlGal1 complex, setting KlGal4 free to activate transcription. This is an interesting article which should be published in Life Science Alliance after the criticisms below have been addressed.
Minor criticism: According to the materials and methods, all fusions expressed from plasmids are under the control of the S. cerevisiae ADH1 promoter. While it would have been better to use the KlGAL80 promoter, the fluorescent microscopic pictures presumably look nicer this way. It is not clear if the KlGal80myc fusions were integrated such that they are expressed under the control of the KlGAL80 promoter or also under the control of the ScADH1 promoter. In order to be more transparent, the authors should state for each construct from which promoter it is expressed in the respective figure legends.

Major criticisms:
The inhibitory effect of the KlGal80K5A/R6A mutant could be due to high protein levels. For the KlGal80-myc3 and KlGal80K5A/R6A-myc3 fusions, the authors should run a Western Blot with cells grown under repressing and inducing conditions in order to determine the relative expression levels.
The authors have only shown that KlGal1 interacts better with wild-type KlGal80 than with the KlGal80K5A/R6A mutant protein. They should set up a competition assay to directly demonstrate their claim that KlGal1 competes better for the wild-type KlGal80-KlGal4 complex as compared to the mutant KlGal80K5A/R6A-KlGal4 complex.
Reviewer #2 (Comments to the Authors (Required)): The core features of GAL regulation show strong conservation, however, species-specific differences in regulation are known to arise due to differences in expression levels, localization and primary sequence of the regulatory proteins: Gal4, Gal80 and Gal1/Gal3. Reinhardt-Tews et al.
show that the N terminal 16 amino acids of Gal80 (Gal80-N16AA) are required for both nuclear localization and Gal1 binding in K.lactis but not in S.cereviciae. The authors recapitulate their previous finding that Gal80 is predominantly nuclear in K.lactis, but not in S.cerevisiae, and utilize their previously established binding, expression and localization assays to place the role of Gal80-N16AA in the overall GAL regulation. The main/novel claim of this study is that Gal80-N16AA interacts with the catalytic center of nuclear Gal1, which in turn destabilizes Gal80-Gal4 interaction. This is a well written manuscript with clear results that propose a potentially interesting mechanism. However, the main claim of the manuscript needs a more thorough validation.

Major concern
The claim for the interaction of Gal80-N16AA with the catalytic site of Gal1 is supported only through in silico model and not the actual structure. It is clear this region is necessary but not sufficient for interaction with Gal1. Thus, mutations in Gal1 that disrupts interaction with Gal80-N16AA, based on their model, are essential to support their claim.
Minor concerns 1. The suppression of Gal80-KR56A mutation by Gal1 mutant that does not interact with wild-type GAL80 but no suppression by the Gal1 catalytic mutant is a non-intuitive result and might be remotely related to the claim of this paper. This should be discussed. 2. Typo on Page 10 "color on 0,2% glucose" 3. In Figure 7 the models are hard to follow

Author Responses Point-by-Point
Reviewer #1: The authors have identified an NLS at the N-terminus of KlGal80, the inhibitor of KlGal4. The authors have fused KlGal80 to GFP and found that while wild-type Gal80 localized to the nucleus, Gal80 with the mutated NLS K5A/R6A was not localized to the nucleus but present everywhere inside the cell. One would expect that the KlGal80K5A/R6A mutant strain was deficient for inhibition of KlGal4, however, the authors have found that it is actually deficient for induction, as the presence of the mutant  We would also like to highlight that the yeast strains expressing 3x-myc tagged versions have been fixed and permeabilized during the staining procedure to allow the respective antibody to enter the cells. Therefore, the cells in the light microscopy image look miserable whereas the cells expressing GFP-Gal80 look healthy. We have now marked the two techniques, immunofluorescence and (GFP) fluorescence, by red and green bars in a new version of Figure 1. In addition, we have modified the 1st Authors' Response to Reviewers September 2, 2020 figure legends of Figures 1, 2, 4 and S1 to S3 to point out more clearly which technique was used in the respective experiments.

Major criticisms:
The inhibitory effect of the KlGal80K5A/R6A mutant could be due to high protein levels. For the KlGal80-myc3 and KlGal80K5A/R6A-myc3 fusions, the authors should run a Western Blot with cells grown under repressing and inducing conditions in order to determine the relative expression levels.
Authors Answer: We show in the revised version by a Western blot (new Fig. 3B) that wild-type and mutant KlGal80p are expressed at comparable levels. Therefore, we can exclude the possibility that the KlGal80K5A/R6A mutant exhibits its inhibitory effect because of elevated protein levels. We now refer to this new data on page 7, which reads as follows "We can exclude that this phenotype is caused by an elevated concentration of the KlGal80-K5A/R6A protein, the Western blot indicated no difference to wild-type KlGal80p (Fig. 3B)".
In  Fig. S5) we conclude that the KR>AA exchange is responsible for the reduced affinity of KlGal80-K5A/R6Ap for GstKlGal1p in vitro.

Major criticisms:
The authors have only shown that KlGal1 interacts better with wild-type KlGal80 than with the Therefore, it was technically not feasible to perform the competition experiment -titration of Gal4p against the preformed wt and mutant Gal1-Gal80 complexes. Using optimized purification protocols for untagged KlGal80p, we were able to confirm the drastically reduced affinity of the K5A/R6A mutant protein for KlGal1p in the presence of Gal and ATP. As mentioned above, the wild type and mutant Gal80 proteins show identical stability profiles and the inability of KlGal80K5A/R6A to bind Gal1p (in the presence of ATP and galactose) is not related to a general destabilization of the protein (see answer above). We now show this data in the new Figure 6C, replacing the previous version (corresponding input controls are shown in the newly created Supplementary Fig. S6). In the new Fig. 6D we have addressed the question raised by the reviewer in a modified form as described in the text on page 13, which reads as follows "Both KlGal80p variants were detected in the GstGal4p pull down fraction and the intensity of the bands representing one or the other KlGal80p variant was neither affected by the presence or absence of galactose and ATP nor by the presence or absence of KlGal1p in the assay (Fig. 6D). Hence the amount of KlGal80p in the bound fraction was only determined by the GstKlGal4p input (Supplementary Fig. S6). We conclude that in this three-component in vitro binding assay KlGal1p cannot effectively compete with GstKlGal4p." Furthermore, we managed to obtain GST-KlGal4-Gal80 complexes using wild type and mutated KlGal80. We have used these preparations to titrate large excess of purified untagged KlGal1 protein, but we have not obtained any evidence for a displacement of a formed KlGal4-KlGal80 complex under any of the tested conditions. We believe that in vitro large differences in affinity exist between the KlGal80-Gal4 (very high affinity) and the KlGal80-KlGal1p (low affinity). These large differences do not allow the displacement of wild type KlGal80 from the Gal4 complex by KlGal1. Therefore, we were not able to test the differences between wild type and mutated KlGal80 in this in vitro setup or to exclude that these differences do not exist. Most importantly we have shown that in this in vivo scenario the basic residues in the N-terminus have a major impact on KlGal4 activation. In a modification of our model we are taking into account the new in vitro data and assume that the mutation has no major influence on the KlGal80-KlGal4 complex. To better explain our kinetic model, which for the time being has to remain of hypothetical nature, the discussion section was extensively rewritten and adjusted.
Reviewer #2: The core features of GAL regulation show strong conservation, however, species-specific differences in regulation are known to arise due to differences in expression levels, localization and primary sequence of the regulatory proteins: Gal4, Gal80 and Gal1/Gal3. Reinhardt-Tews et al. show that the N terminal 16 amino acids of Gal80 (Gal80-N16AA) are required for both nuclear localization and Gal1 binding in K. lactis but not in S. cerevisiae. The authors recapitulate their previous finding that Gal80 is predominantly nuclear in K. lactis, but not in S. cerevisiae, and utilize their previously established binding, expression and localization assays to place the role of Gal80-N16AA in the overall GAL regulation. The main/novel claim of this study is that Gal80-N16AA interacts with the catalytic center of nuclear Gal1, which in turn destabilizes Gal80-Gal4 interaction. This is a well written manuscript with clear results that propose a potentially interesting mechanism. However, the main claim of the manuscript needs a more thorough validation.

Major concern
The claim for the interaction of Gal80-N16AA with the catalytic site of Gal1 is supported only through in silico model and not the actual structure. It is clear this region is necessary but not sufficient for interaction with Gal1. Thus, mutations in Gal1 that disrupts interaction with Gal80-N16AA, based on their model, are essential to support their claim.
Authors Answer: If the mutation disrupts a specific amino acid interaction of N16AA with KlGal1p that is necessary for KlGal4p activation, there may be Klgal1 mutants that have a suppressor phenotype because the interaction is restored. We have tried to obtain such mutants by mutagenesis of KlGAL1 screening for mutants that suppress the KlGAL80-K5A/R6A mutation. We have obtained and further characterized 8 such mutants. All but one had multiple (2-4) amino acid exchanges, three of them had identical double mutations. Six of the amino acid substitutions were introduced as single mutations into the KlGAL1 WT gene. Among them two are of major interest by affecting specifically the Klgal80-K5A/R6A mutant but not the KlGAL80 WT allele. We provide a confidential cartoon where these residues are marked, specifically for the reviewers. For us, the positions of the mutations did not reveal any candidate with the desired properties. Hence, we decided not to include these data in the present manuscript. A further characterization of the mutants is for sure required, but beyond the scope of this report.

Minor concerns
1. The suppression of Gal80-KR56A mutation by Gal1 mutant that does not interact with wild-type GAL80 but no suppression by the Gal1 catalytic mutant is a non-intuitive result and might be remotely related to the claim of this paper. This should be discussed.
Authors Answer: We thank the reviewer for this stimulating comment. We had a closer look at the Klgal1 mutants that had been described and came up with a kinetic model to explain their various phenotypes. Intriguingly, the proposed mode of regulation would also explain a number of findings in the literature that cannot easily be reconciled with the current dissociation models. This model is supported by the new biochemical data shown in the new version of Figure 6D and the genetic data shown in Figure 5.  Thank you for submitting your revised manuscript entitled "A double role of the Gal80 N-terminus in activation of transcription by Gal4p". We would be happy to publish your paper in Life Science Alliance (LSA) pending final revisions necessary to meet our formatting guidelines.
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