AtTPR10 Containing Multiple ANK and TPR Domains Exhibits Chaperone Activity and Heat-Shock Dependent Structural Switching

Round 1

Reviewer 1 Report

Summary

This work identifies a novel function of the Arabidopsis thaliana tetratricopeptide repeat protein AtTPR10 as a molecular chaperone. Investigational interest in AtTPR10 is sparked by its peculiar structure containing both TPR and ANK putative binding domains. The authors underline the significance of their findings as being instrumental for the development of stress-tolerant crops. AtPR10 heat stability, structural shifts favourable for chaperone activity and temperature dependent expression and activity are convincingly showed through a series of diverse and complementary experiments. A portion of these experiments are repeated on differentially truncated proteins and demonstrate that the two binding domains (TRP and ANK) synergistically contribute to AtTPR10 chaperone activity.

Major concerns

None

Minor Concerns

Where no data average is reported in figures, reproducibility of that particular finding cannot be appreciated. Please specify whether the displayed data is a representative result of multiple repeats of the same experiments and if so, of how many repeats. This applies to the majority of figures except for 4b, 7c and 8a, as suggested by the presence of error bars. “Homogeneous AtTPR10 purified as a single band on a SDS-PAGE was subjected to native gel electrophoresis”. This implies that AtTPR10 has been renaturated following SDS-PAGE in order to subject it to native gel electrophoresis, is that correct? If so, please indicate method and conditions that were used to refold the denaturated protein. Please indicate kDa size in SDS-PAGE image in figures 5a and 9a. “The heat shock-dependent formation of HMW complexes suggests that the AtTPR10 protein interacts with itself and forms higher MW complexes by hydrophobic interaction.” Please clarify how does it suggest that binding occurs specifically through hydrophobic interaction? Figure 7d: Please indicate the incubation temperature used in this experiment as well as what constitutes the control condition.

Author Response

Response to Reviewer 1 Comments

Point 1: Where no data average is reported in figures, reproducibility of that particular finding cannot be appreciated. Please specify whether the displayed data is a representative result of multiple repeats of the same experiments and if so, of how many repeats. This applies to the majority of figures except for 4b, 7c and 8a, as suggested by the presence of error bars.

Response 1: In fact, all the experiments have been done at least three biological independent replicates. Based on your comment, we clearly specified the reproducibility of our data by adding the sentence “the representative results are means of at least three independent experiments” in the revised manuscript and “The data presented are the averages of at least three independent measurements” in revised figure legends, line 214, 306, and 322.

 

Point 2: “Homogeneous AtTPR10 purified as a single band on a SDS-PAGE was subjected to native gel electrophoresis”. This implies that AtTPR10 has been renaturated following SDS-PAGE in order to subject it to native gel electrophoresis, is that correct? If so, please indicate method and conditions that were used to refold the denaturated protein.

Response 2: No, we have prepared several protein samples and analyzed their properties at the same times. Thus, after confirming the purity of AtTPR10 as a single band on a SDS-PAGE gel, we analyzed its protein structure by using another aliquot of AtTPR10 on native gel electrophoresis. The sentence and experimental procedures were clearly revised in the lines 217-218, page 7 and in the revised manuscript.

 

Point 3: Please indicate kDa size in SDS-PAGE image in figures 5a and 9a.

Response 3: Based on your comment, we clearly indicated the ‘kDa’ in SDS-PAGE image in figures 5a and 9a (line 239 and line 335).

 

Point 4: “The heat shock-dependent formation of HMW complexes suggests that the AtTPR10 protein interacts with itself and forms higher MW complexes by hydrophobic interaction.” Please clarify how does it suggest that binding occurs specifically through hydrophobic interaction?

Response 4: Thanks for your critical comment. As you indicated, it is not possible to define that the binding force of AtTPR10 to form heat shock-dependent formation of HMW complexes is specifically attributed through the hydrophobic interaction. However, since heat shock makes the exposure of hydrophobic domain of proteins, it should be reasonable to describe that hydrophobicity might be a main factor to form HMW complexes.

Based on your comment, we revised the sentence from “The heat shock-dependent formation of HMW complexes suggests that the AtTPR10 protein interacts with itself and forms higher MW complexes by hydrophobic interaction” to “The heat shock-dependent formation of HMW complexes suggests that the AtTPR10 protein interacts with itself and forms higher MW complexes mainly by hydrophobic interaction” in the line 230, page 7.

 

Point 5: Figure 7d, Please indicate the incubation temperature used in this experiment as well as what constitutes the control condition.

Response 5: According to your comment, we clearly indicated the incubation temperature used in this experiment and control conditions in the legend of figure 7 in line 308 and line 309.

 

Please see the attachment, thanks

Author Response File: Author Response.docx

Reviewer 2 Report

In this manuscript, the authors identified biochemical function of AtTPR10 protein which contains three TPR domain repeats at the C-terminus and seven ankyrin (ANK) domain repeats at the N-terminus using DU800 spectrophotometer, size exclusion chromatography, SFM25 spectrofluorometer, RT-PCR, SDS PAGE and Native PAGE. The researchers found that AtTPR10 functions as a molecular chaperone to protect intracellular proteins from thermal stresses and both TPR and ANK repeats show each domain displays a similar chaperone activity.

The research topic is interesting, and the study is technically sound. It includes evidence for each findings at certain level.

The authors mostly provided enough experimental details that the experiments could be reproduced but experimental conditions for native gel electrophoresis are not provided. P7, Lane 217- Incubation time for the protein is given as 20 minutes but it is stated as 30 min at page 8, Lane 234-236, Figure 5a. Clarification is needed.

Overall, I very much enjoyed reading this manuscript and recommend its publication.

Author Response

Response to Reviewer 2 Comments

 

Point 1: The authors mostly provided enough experimental details that the experiments could be reproduced but experimental conditions for native gel electrophoresis are not provided.

Response 1: Based on your comment, we provide the experimental conditions for native gel electrophoresis in detail, as shown in line 137-144, page, 4.

 

Point 2: P7, Lane 217- Incubation time for the protein is given as 20 minutes but it is stated as 30 min at page 8, Lane 234-236, Figure 5a. Clarification is needed.

Response 2: Thank for pointing our mistake. Considering your comment, we clearly revised the reaction conditions at line 223, page 7.

 

Please see the attachment, thanks

Author Response File: Author Response.docx