TMX5/TXNDC15, a natural trapping mutant of the PDI family is a client of the proteostatic factor ERp44

  1. Tatiana Soldà
  2. Carmela Galli
  3. Concetta Guerra
  4. Carolin Hoefner
  5. Maurizio Molinari

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Abstract

The endoplasmic reticulum (ER) is the organelle of nucleated cells that produces lipids, sugars and proteins. More than 20 ER-resident members of the Protein Disulfide Isomerase (PDI) family regulate formation, isomerization and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4 and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multi-pass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57 and ERp44. The association of TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER retention of TMX5. Thus, TMX5 belongs to the family of proteins including Ero1α, Ero1β, Prx4, ERAP1, SUMF1 that do not display ER retention sequences and rely on ERp44 engagement for proper inter-compartmental distribution.

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    Manuscript number: RC-2024-02555

    __Corresponding author(s): __Maurizio Molinari

    [Please use this template only if the submitted manuscript should be considered by the affiliate journal as a full revision in response to the points raised by the reviewers.

    If you wish to submit a preliminary revision with a revision plan, please use our "Revision Plan" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

    1. General Statements [optional]

    We thank the 3 reviewers for the positive and constructive comments to our manuscript.

    Please see below the point-by-point responses to their suggestions.

    2. Point-by-point description of the revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The paper proposes an interesting role for ERp44 in TMX5 retention. The authors identified a list of proposed TMX5 clients which include many Golgi localised proteins but do not discuss the role for TMX for instance in protein folding. In this context it is not absolutely clear whether TMX5 acts as a trafficking chaperone? are clients functionally engaged in the Golgi or ER or both?

    This work focuses on the crosstalk between a member of the PDI superfamily lacking a conventional cytosolic ER retention motif (TMX5), and ERp44, a PDI family member previously reported to retrieve in the ER proteins that lack the ER retention motif (ERp44). To support our conclusions on the involvement of ERp44 in control of TMX5’s intracellular distribution, we have added new data obtained by characterization of a cell line lacking ERp44, where more than 50% of TMX5 escapes ER retention (new Fig. 6).

    __We agree with the referee that the assessment of the biological role of TMX5 is of interest. We mention in this manuscript that there is a follow-up study (ongoing in the lab) on TMX5 clients and TMX5 function. More specifically, we are monitoring the action of TMX5 on the biogenesis and intracellular trafficking of class I HLA molecules, which are, besides PDI, ERp57 and ERp44, major interactors (clients) of TMX5 (please also refer to the initial and final parts of the new discussion). __

    The defining criteria for the client proteins were not included. At last, it might be of interest to evaluate for how long TMX5 clients are retained on the protein, whether it is temporary (as for instance a folding sensor) or more permanent.

    The list of interacting proteins is now available (Data are available via ProteomeXchange with identifier PXD054716."), their selection for presentation in Figure 3B is now explained more clearly (Results, page 6). Also better explained is that we define as “clients of TMX5” those endogenous proteins that associate with TMX5, covalently, via the catalytic Cys220. The mutation of the TMX5 active site cysteine residue does not impact the covalent association of PDI, ERp57 and ERp44 with TMX5. For this reason, we do not consider these PDI family members clients of TMX5. In this submission, we explore the covalent association of ERp44 and its consequences on TMX5 subcellular distribution. Interacting via non-catalytic Cysteine residues 114 and 124 with the catalytic cysteine 29 of ERp44, we identify TMX5 as a client of the latter.

    The preparation of figures could be greatly improved and there is some inconsistency among similar gels.

    Please refer to the point-by-point answers below.

    The proposed model of ERp44, ER retention vs ER retrieval, is unclear. Overall, there is more room for improved discussion beyond the conclusions from experiments.

    __We thank the referee for these comments. We have improved the description of the results, and we separated the Discussion (written de novo) from the Results section. __

    The ERp44 interaction is interesting especially since the protein contains an incomplete thioredoxin domain (such as ERp29, PDIA17 and 18), would the interaction between Erp44 and TMX5 be involved in some holdase/competitor role thereby allowing for client selectivity (or kinetics)? In addition, all the experiments were carried out in Hek293T or MEF cells, would the authors anticipate some interactions of TMX5 with PDIA17/18 in cells where those proteins are highly expressed? Testing whether the observation is a general mechanism occurring between TMX5 and PDI family members with incomplete thioredoxin sites would be an asset.

    __We thank the referee for this comment that we implemented in the new discussion. __

    Major comments Fig 2

    • avoid labels on the blots that might obscure information and impede clarity and interpretation. o The % of resistant protein can be otherwise placed.

    __This has been modified, thank you. __

    • What does the asterix in 2B signify? This should be included in the legend.

    We have now specified in the legend of figures that asterisks show cross-reacting polypeptide bands.

    • A label for 'deglycosylated' proteins could be included.

    __We added a label for de- and for glycosylated proteins in the EndoH essays in Figs. 2A, 2B and 5B. __

    • Consider treating with PNGase.

    This is now showy in panel 2A, lane 5.

    • There is a change in EndoH resistance of about 3-4% among wt, C220A & C114A, is this significant?

    We do not consider significant these variations. Our data show that the mutation of TMX5 Cys 114 or of Cys124 to alanine substantially reduce (without abolishing) the co-IP with ERp44. This means that the proteins interact less, or that the interaction is more short living. The EndoH experiment shown in Fig. 2B and the CLSM analyses in Figs. 2D-2O fail to reveal significant differences showing that these reduced or more short living associations with ERp44 are sufficient to control TMX5 distribution.

    In the previous submission, the function of ERp44 in retaining TMX5 in the ER was supported by data showing that the co-expression of ERp44 retains TMX5 in the ER, but co-expression of ERp44C29S that cannot bind TMX5 fails to retain TMX5 in the ER. These model is further supported in this new submission by the release of 50% of TMX5 from the ER in cells lacking ERp44, which is substantially inhibited to the levels measured in wild type cells upon back-transfection of ERp44, but not upon the co-transfection of the ERp44C29S mutant (new Figure 6).

    • Equivalent inputs (cell lysates) for the IPs should be included.

    __ These have now been added in Figs. 4, 5 6, and 7.__

    Fig 3A

    • Indicate the specific bands that were subject to MS. How did the authors correct for non-specific interactors and false positives? Perhaps a more specifically targeted approach could be utilised.

    • How do the authors explain the absence of bands representing the reduced form of interacting TMX5 interactors?

    • What was the inclusion and exclusion criteria used to determine which of the proteins listed were clients?

    The endogenous proteins present in the entire region of the gel labeled with the red and blue rectangles have been sequenced (see methods section and this is now also better explained in the results section, page 6). Only the proteins that disappear from the corresponding region of the gel when the samples have been reduced are listed in the table. This is also better explained in the text (page 6). These experiments have been repeated few times with a series of controls (e.g., mock-transfected cells and cells transfected with other members of the TMX family (shown to capture and to impact on the fate of other endogenous polypeptides in previous publications from our lab)). An in parallel analysis of mock, TMX3, TMX4 and TMX5 interactors has been published in (Kucinska et al Nature Comm 2023), where we focused on the biological function of TMX4. The references referring to the TMX1 study (Brambilla et al 2015) and the TMX4 study (Kucinska et al) are given in the text.

    The Table in Fig. 3B only lists the interacting polypeptides that have a MW __- It might be useful to perform MS on the C220A mutant and compare those results to the WT.

    __To validate few interactions with endogenous proteins detected in MS, and to compare the interactions of TMX5 and TMX5C220A, we have used the specifically targeted approach suggested above by the referee (i.e., co-IP validated by WB, Figs. 3C-3F).

    __

    Fig 4

    • Equivalent inputs (cell lysates) for the IPs should be included.

    __This is now shown as panel A in Fig. 4.____

    __

    Fig 5

    • Equivalent inputs (cell lysates) for the IPs should be included.

    This is now Figure 7, see new panel 7A

    Fig 6

    • A loading control should be included.

    __This is now Fig. 5. Both panels A and B in Fig. 5 show total cell extracts____

    __

    • Blot using anti-HA to identify ERp44 should be included to substantiate claims.

    The ERp44 and TMX5 components of the ERp44-s-s-TMX5 mixed disulfides are detected upon IP:HA followed by WB:V5 (to show the TMX5 component) and upon IP:V5 followed by WB:HA to show the ERp44 component) in Figs. 4B-4E and 7B-7C.

    • How do the authors account for the huge difference in TMX5 associated complexes shown in Fig 6A compared to Fig 3A.

    Fig. 6 is now Fig. 5. As specified in the legends of the figures, Fig. 3A shows a gel, where the complexes are stained with silver, Fig. 5A is a WB, where the complexes are stained with an antibody. The intensities of the signals cannot be compared.

    • Inappropriate marking on the gel area.
    • Inconsistencies in protein standard labeling

    This has carefully been checked and corrected where needed. Please note that we used two different MW standards for our figures (200, 117, 97, 66, 45, 31 kDa and 270, 175, 130, 95, 66, 53, 37 kDa)

    • It might be useful to demonstrate the colocalisation of ERp44 and ERp44C29S with Giantin and with TMX5 considering that ERp44 is known to cycle between the Golgi and ER.

    These data are shown in Fig. 5D-5I.

    Reviewer #1 (Significance (Required)):

    This work provides an additional understanding on how the regulation of Erp44 trafficking might occur (and perhaps additional PDIs), and lead to the characterization of kinetic value that might explain better productive protein folding in the early secretory pathway. This represents a significant advance in the field and may in turn unveil uncharacterized pathophysiological functions in various diseases. This is a serious study well conducted and original by an expert in the field that desserves publication.

    Field of expertise: ER homeostasis control

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Solda et al, investigates TMX5, a poorly understood member of the PDI family that lacks an ER-retrieval motif. They find that it localizes to the ER and the Golgi and that it interacts with ERp44. This interaction requires formation of a mixed disulfide and they identify the cysteine residues in both proteins that mediate this interaction. Overall, this is a well written manuscript that is easy to follow and the story is compact and straight-forward. It provides some new and solid insight into the biology of TMX5 without going into depth of what the cellular role of TMX5 is or might be. I have only very few comments and suggestions:

    1- The authors conclude that ERp44 associates only with ER-localized TMX5. I am not sure that this is a valid conclusion based on the data. EndoH sensitivity just means that the protein has not gone to the medial Golgi. The pool of TMX5 could therefore be an ERGIC-based pool, or it could interact with a TMX5 that is recycled directly from the first Golgi cisterna, where complex glycosylation is unlikely to occur. Can this be validated using another type of experiment? Alternatively, the wording could be changed.

    We thank the reviewer. We agree with this insightful comment that led us to change the wording used in some part of the text.

    2- Is the trafficking of TMX5 dependent on its glycosylation?

    This is another insightful comment that we report in the ____new discussion, where we write, page 14 “____It should be noted that in the case of TMX5 the extensive N-glycosylation could engage____ leguminous L-type lectins located in the ER (VIPL), cycling between the ER and the intermediate compartment (ERGIC) (ERGIC-53) or between the ERGIC and the cis-Golgi (VIP36)____33-36____ and have an impact on the subcellular distribution and activity of TMX5.____”

    3- Figure 6: The data are not really convincing. Just because the color turns yellow, it does not mean that there is colocalization. The green channel is overexposed in this area of the cell, and anything will produce a yellow color, even if there is no genuine colocalization. Maybe the authors could provide a different example and even better would be a quantification of the colocalization.

    __We thank the referee for this comment. We show images of better quality, where the black/white channels clearly show the co-localization (or lack thereof) of TMX5 with the Golgi marker Giantin in cells mock-transfected (co-localization TMX5:Giantin, Fig. 5D), co-transfected with ERp44 (no co-localization TMX5:Giantin, Fig. 5E), or co-transfected with ERp44C29S, co-localization TMX5:Giantin, Fig. 5F). Figs. 5G-5I show the corresponding results for the co-localization or lack thereof between TMX5C220A and Giantin. Importantly, the IF data match the data shown in Fig. 5B, where release from the ER (or arrival in the medial Golgi, see text of the manuscript and comment 1 by the referee) is assessed by monitoring complex glycosylation. __

    Reviewer #2 (Significance (Required)):

    This is a solid story that will be of interest of scientists working on various aspects of the secretory pathway and protein quality control. The advance is rather incremental, because there are no experiments that provide insight into the cellular roles of TMX5.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Solda et al have assembled data on the transmembrane redox enzyme TMX5, on which currently very little information is available. TMX5 does not contain any obvious targeting signal, unlike the other TMX family proteins, which localize to the ER. TMX5 has 5 glycosylation sites, which can be used to determine its intracellular localization biochemically. Indeed, about 20% of TMX5 is found as endoH-resistant, indicating Golgi localization. This is confirmed with beautiful IF imagery and giantin co-localization. The Golgi localization requires two luminal cysteines (C212, 177), which likely form a disulfide bond. TMX5 acts as a natural cysteine-trapping protein, allowing for easy assessment of its interactors. Within its interactome, the authors found multiple members of the thioredoxin family. Many of these interactions occur within the CXXS motif, but notably ERp44 does not require this motif to interact, indicating this and other interactions are not of a catalytic nature. Instead, the authors found this interaction to be essential for ER retention or retrieval and depends on the cysteine within the ERp44 "active" site. The study provides critical first insight about the potential functions and sites of activity of TMX5.

    Specific Points:

    1. The results are very convincing and of high quality.
    2. The cytosolic tail of TMX5 contains an LI motif, which could act as a post-ER localization signal. Since the protein might play a role in ciliogenesis, this motif could be critical. In this context, I am wondering which mutations are known to lead to the disease spectrum.

    The position of disease-related TMX5 mutations identified so far are given in Xu H, et al (2024) Mol Genet Genomic Med 12: e2340 ____https://www.ncbi.nlm.nih.gov/pubmed/38073519____ and in ____Deng T, Xie Y (2024) Mol Genet Genomic Med 12: e2343 https://www.ncbi.nlm.nih.gov/pubmed/38156946____.

    They are all distributed in the luminal part of the protein____.

    Mutation of C114 and C124 abrogates interaction with ERp44. Therefore, I would expect these mutations to increase endoH resistance and Golgi staining. This should be investigated by the authors.

    __The mutations C114 and C124 reduce (or make short-living), without abrogating the covalent association between TMX5 and ERp44. The EndoH experiment shown in Fig. 2B and the IF in Figs. 2D-2O fail to reveal significant differences showing that these reduced or more short living associations with ERp44 are sufficient to control TMX5 distribution. To strengthen our conclusion that ERp44 is involved in regulation of the intracellular TMX5 distribution, we have now added data in ERp44 cell (50% of TMX5 displays complex glycans as symptom of traffic to the medial Golgi compartment), back-transfection of ERp44 (but not of the ERp44C29S mutant that does not associate with TMX5) restores the complex glycan fraction to the level measured in wild type cells (Fig. 6). __

    Minor Points:

    1. The position of the % endoH resistance in Figures 1B and 6B is not ideal, as it obstructs a visual inspection of TMX5 resistance to endoH.

    This has been modified, thank you.

    Reviewer #3 (Significance (Required)):

    Given that no information about TMX5 is currently available, the study provides critical first insight that should allow researchers to tackle the disease relevance of TMX5 in the future.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    Solda et al have assembled data on the transmembrane redox enzyme TMX5, on which currently very little information is available. TMX5 does not contain any obvious targeting signal, unlike the other TMX family proteins, which localize to the ER. TMX5 has 5 glycosylation sites, which can be used to determine its intracellular localization biochemically. Indeed, about 20% of TMX5 is found as endoH-resistant, indicating Golgi localization. This is confirmed with beautiful IF imagery and giantin co-localization. The Golgi localization requires two luminal cysteines (C212, 177), which likely form a disulfide bond. TMX5 acts as a natural cysteine-trapping protein, allowing for easy assessment of its interactors. Within its interactome, the authors found multiple members of the thioredoxin family. Many of these interactions occur within the CXXS motif, but notably ERp44 does not require this motif to interact, indicating this and other interactions are not of a catalytic nature. Instead, the authors found this interaction to be essential for ER retention or retrieval and depends on the cysteine within the ERp44 "active" site. The study provides critical first insight about the potential functions and sites of activity of TMX5.

    Specific Points:

    1. The results are very convincing and of high quality.
    2. The cytosolic tail of TMX5 contains an LI motif, which could act as a post-ER localization signal. Since the protein might play a role in ciliogenesis, this motif could be critical. In this context, I am wondering which mutations are known to lead to the disease spectrum.
    3. Mutation of C114 and C124 abrogates interaction with ERp44. Therefore, I would expect these mutations to increase endoH resistance and Golgi staining. This should be investigated by the authors.

    Minor Points:

    1. The position of the % endoH resistance in Figures 1B and 6B is not ideal, as it obstructs a visual inspection of TMX5 resistance to endoH.

    Significance

    Given that no information about TMX5 is currently available, the study provides critical first insight that should allow researchers to tackle the disease relevance of TMX5 in the future.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Solda et al, investigates TMX5, a poorly understood member of the PDI family that lacks an ER-retrieval motif. They find that it localizes to the ER and the Golgi and that it interacts with ERp44. This interaction requires formation of a mixed disulfide and they identify the cysteine residues in both proteins that mediate this interaction.

    Overall, this is a well written manuscript that is easy to follow and the story is compact and straight-forward. It provides some new and solid insight into the biology of TMX5 without going into depth of what the cellular role of TMX5 is or might be. I have only very few comments and suggestions:

    1. The authors conclude that ERp44 associates only with ER-localized TMX5. I am not sure that this is a valid conclusion based on the data. EndoH sensitivity just means that the protein has not gone to the medial Golgi. The pool of TMX5 could therefore be an ERGIC-based pool, or it could interact with a TMX5 that is recycled directly from the first Golgi cisterna, where complex glycosylation is unlikely to occur. Can this be validated using another type of experiment? Alternatively, the wording could be changed.
    2. Is the trafficking of TMX5 dependent on its glycosylation?
    3. Figure 6: The data are not really convincing. Just because the color turns yellow, it does not mean that there is colocalization. The green channel is overexposed in this area of the cell, and anything will produce a yellow color, even if there is no genuine colocalization. Maybe the authors could provide a different example and even better would be a quantification of the colocalization.

    Significance

    This is a solid story that will be of interest of scientists working on various aspects of the secretory pathway and protein quality control. The advance is rather incremental, because thereare no experiments that provide insight into the cellular roles of TMX5.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The paper proposes an interesting role for ERp44 in TMX5 retention. The authors identified a list of proposed TMX5 clients which include many Golgi localised proteins but do not discuss the role for TMX for instance in protein folding. In this context it is not absolutely clear whether TMX5 acts as a trafficking chaperone? are clients functionally engaged in the Golgi or ER or both? The defining criteria for the client proteins were not included. At last, it might be of interest to evaluate for how long TMX5 clients are retained on the protein, whether it is temporary (as for instance a folding sensor) or more permanent.

    The preparation of figures could be greatly improved and there is some inconsistency among similar gels. The proposed model of ERp44, ER retention vs ER retrieval, is unclear. Overall, there is more room for improved discussion beyond the conclusions from experiments.

    The ERp44 interaction is interesting especially since the protein contains an incomplete thioredoxin domain (such as ERp29, PDIA17 and 18), would the interaction between Erp44 and TMX5 be involved in some holdase/competitor role thereby allowing for client selectivity (or kinetics)? In addition, all the experiments were carried out in Hek293T or MEF cells, would the authors anticipate some interactions of TMX5 with PDIA17/18 in cells where those proteins are highly expressed? Testing whether the observation is a general mechanism occurring between TMX5 and PDI family members with incomplete thioredoxin sites would be an asset.

    Major comments

    Fig 2

    • avoid labels on the blots that might obscure information and impede clarity and interpretation.
    • The % of resistant protein can be otherwise placed.
    • What does the asterix in 2B signify? This should be included in the legend.
    • A label for 'deglycosylated' proteins could be included.
    • Consider treating with PNGase to .....
    • There is a change in EndoH resistance of about 3-4% among wt, C220A & C114A, is this significant?
    • Equivalent inputs (cell lysates) for the IPs should be included.

    Fig 3A

    • Indicate the specific bands that were subject to MS. How did the authors correct for non-specific interactors and false positives? Perhaps a more specifically targeted approach could be utilised.
    • How do the authors explain the absence of bands representing the reduced form of interacting TMX5 interactors?
    • What was the inclusion and exclusion criteria used to determine which of the proteins listed were clients?
    • It might be useful to perform MS on the C220A mutant and compare those results to the WT.

    Fig 4

    • Equivalent inputs (cell lysates) for the IPs should be included.

    Fig 5

    • Equivalent inputs (cell lysates) for the IPs should be included.

    Fig 6

    • A loading control should be included.
    • Blot using anti-HA to identify ERp44 should be included to substantiate claims.
    • How do the authors account for the huge difference in TMX5 associated complexes shown in Fig 6A compared to Fig 3A.
    • Inappropriate marking on the gel area.
    • Inconsistencies in protein standard labeling
    • It might be useful to demonstrate the colocalisation of ERp44 and ERp44C29S with Giantin and with TMX5 considering that ERp44 is known to cycle between the Golgi and ER.

    Significance

    This work provides an additional understanding on how the regulation of Erp44 trafficking might occur (and perhaps additional PDIs), and lead to the characterization of kinetic value that might explain better productive protein folding in the early secretory pathway. This represents a significant advance in the field and may in turn unveil uncharacterized pathophysiological functions in various diseases.

    This is a serious study well conducted and original by an expert in the field that desserves publication.

    Field of expertise: ER homeostasis control

  5. Version published to 10.1101/2024.06.12.598629v1 on bioRxiv