The San1 Ubiquitin Ligase Avidly Recognizes Misfolded Proteins through Multiple Substrate Binding Sites
Round 1
Reviewer 1 Report
The yeast San1 is one of the ubiquitin ligases for protein quality control (PQC). Besides having a catalytic RING-finger domain, San1 contains long stretches of disordered regions. However, the precise mechanism how San1 recognizes its substrates via these disordered regions remains to be elucidated. In this manuscript, the authors reconstituted the San1 ubiquitination reaction system using fully recombinant proteins and synthesized substrate peptides. Their experimental designs are interesting, but there might be problems with the designs and the interpretation of the results.
- In figure 2, the authors hypothesized that if the sites of proteolysis overlap with substrate binding regions, the addition of substrate may protect San1 from proteolysis. However, for this hypothesis, the authors should confirm that San1 peptide substrate and misfolded luciferase are not substrates for chymotrypsin and trypsin and are not digested with these enzymes.
- In figure 3, the authors should increase the concentration of unlabeled San1 peptide until saturation is achieved and confirm that saturation is observed at lower concentration in San11-303 than in San1FL because the authors speculate that some of the binding sites for the peptide substrate are deleted in San11-303. In the present data, although ubiquitination level was decreased in San11-303 even at the lowest concentration of unlabeled San1 peptide, unlabeled San1 peptide did not cause competitive inhibition at all concentrations, so this reviewer cannot judge whether their speculation is reasonable or not.
- It would be better to conduct the experiments in Figure 4 over longer time scales. During single encounter ubiquitination reactions, the amounts of lower ubiquitinated species like mono- and di-ubiquitinated ones should be decreased at longer time points in contrast to multi-turnover ubiquitination, which is not observed in the present Figure 4.
- There is no example to show the binding with weak affinity. The definition of “very high/exceptionally strong” affinity is unclear in Figure 5.
- Related to the comment No. 2, it is unclear whether unlabeled competitor peptide substrate was added enough to be saturated in Figure 6A.
- It should be confirmed whether the san11-303 mutant proteins are as stable as the wild-type ones in san1Δ yeast cells.
Author Response
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Author Response.pdf
Reviewer 2 Report
This paper present biochemical experiments that convincingly show that the San1 ubiquitin ligase, an important component of the protein quality control (PQC) system, have multiple binding sites that bind with high affinity to misfolded substrates. The results could be replicated with a novel C-terminal truncation construct that shows higher purity and homogeneity than full length San1 but has qualitatively similar results and a lower fraction of ubiquitinated substrate that suggest that some substrate binding sites may have been deleted by the truncation. The result also support some level of substrate specificity of the binding sites, although more detailed experiments, and in particular structural experiments, are necessary for better characterizing this interesting system.
The paper is well presented and the conclusions are sound. I only have very minor concerns:
L.68: "San1 substrate binding sites may each recognize a diverse cohort of misfolded protein substrates with little or even no substrate specificity". The word "diverse" seems to contrast with the hypothesis that there is no substrate specificity, please reformulate.
Figure 2: the two curves are difficult to distinguish, please use different line symbols.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The revised manuscript by Jiang and colleagues is improved over the original manuscript. It is obvious that the authors tried to address the criticisms that were made during the first review. However, I am not fully satisfied with a response to my comments described below.
While we understand the reviewer's concern, the suggested experiment is nontrivial since there are two pools of substrate in the reactions: free substrate and substrate bound to San1. Since it is reasonable to suspect that the binding of either peptide substrate or misfolded luciferase to San1 will result in the protection of both trypsin and chymotrypsin sites, it would be extremely difficult to differentiate free versus bound populations. As such, even if substrates in the absence of San1 are proteolyzed in the presence of trypsin and/or chymotrypsin, this likely is not indicative of what happens when these substrates are bound to San1.
Nevertheless, it may be the case that proteolysis of free substrate may diminish the degree of protection observed by substrate binding to San1 which is now discussed in the manuscript (please see lines 291-3).
This reviewer just worries that peptide substrate and misfolded luciferase will inhibit digestion of San1 by trypsin and chymotrypsin when peptide substrate and misfolded luciferase are substrates for trypsin and chymotrypsin, irrespective of whether these substrates are in a free or substrate-bound form. Peptide substrate and misfolded luciferase will be competitive inhibitors of trypsin and chymotrypsin to proteolyze San1.
Author Response
please see the attachement
Author Response File: Author Response.pdf
