An unconventional interaction interface between the peroxisomal targeting factor Pex5 and Eci1 enables PTS1 independent import

  1. Lior Peer
  2. Nadav Elad
  3. Orly Dym
  4. Asa Tirosh
  5. Jossef Jacobovitch
  6. Shira Albeck
  7. Maya Schuldiner
  8. Yoav Peleg
  9. Einat Zalckvar

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Accurate and regulated protein targeting to organelles is crucial for eukaryotic cellular function and homeostasis. This has driven the evolution of targeting signals on proteins and the targeting factors that recognize them. One example for this is peroxisomal matrix proteins, the majority of which rely on the targeting factor Pex5 to correctly localize and function. While most Pex5 cargos contain a Peroxisomal Targeting Signal type 1 (PTS1), in recent years it has become clear that more binding interfaces exist, and that targeting by Pex5 is more complex than previously thought. Here, we uncover that the matrix protein Eci1 can reach peroxisomes in the absence of its PTS1. By solving the structure of a complex between full length yeast Pex5 and Eci1 using Cryo-Electron Microscopy, we could identify their binding interfaces. This allowed us to map an additional binding interface that is independent of the canonical PTS1-mediated binding site. Our work brings forward a solution to a long-standing mystery regarding Eci1 targeting to peroxisomes. More globally, it demonstrates the intricate and complex nature of organelle targeting and how it has evolved to serve the complex eukaryotic environment.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time.

  2. 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 #3

    Evidence, reproducibility and clarity

    Summary: It has been known for many years that some peroxisomal proteins are imported by the major peroxisomal protein import receptor Pex5, which recognises the C terminal targeting signal PTS1, despite either lacking a PTS1 or if the PTS1 is blocked. Some proteins are also able to 'piggyback' into peroxisomes by binding to a partner which possesses a PTS. Eci1, the subject of this study is such a protein. This manuscript identified a PTS1-independent, non-canonical interaction interface between S. cerevisiae PEX5 and imported protein Eci1. Confocal imaging was used to observe the PTS1-independent import of Eci1 into peroxisomes and to establish dependence of Pex5 even in the absence of its piggyback partner Dci1. The authors purified the Pex5-Eci1 complex and used Cryo-EM to provide a structure of the purified PEX5-Eci1 complex. In general, this manuscript is well written and easy to read.

    Major points

    Most of the experiments presented are well-designed and accompanied with appropriate controls. However, please mention how many times the experiments have been repeated and how many biological samples were used in the analysis.The authors should also consider the following suggestions substantiate their conclusions:

    Figure 1A: Include full-length Eci1 with an N-terminal fluorophore, Eci1 PTS1-deletion with N-terminal fluorophore, and the PTS1 deletion with a C-terminal fluorophore, to control for any disturbance of targeting by the C terminal NG tag.

    Figure 1C: Confirm the Eci1 and Dci1 levels (if an antibody is available for the latter) by western blot. It is difficult to compare expression levels when comparing just a small number of cells in the microscope. Western blot would give a more robust evaluation of protein levels and help corroborate the claim that Eci1 expression is decreased in the absence of Dci1 if the authors wish to stand by this conclusion.

    Figure 2: confirm the deletion and overexpression of PEX9, PEX5, and PEX7 by western blot of the relevant strains. The production of these strains is not described in the manuscript. If they have been previously described this should be referenced if not it should be included.

    Figure 2: Validate these strains by checking import of a canonical PTS1 and canonical PTS2 and pex9 dependent protein to ensure they function as they should, unless these strains have been published elsewhere in which case their characterisation can be referenced.

    Figure 3: The gel should include a standard of a known amount of the lysate used in the pull down to enable a semi-quantitative estimation of the amount of Eci1 protein captured by PEX5 with and without its PTS1. Also include Eci1 with a C-terminal fluorophore to be comparable with the in vivo data in Figs 1 and 2. A control with no pex5 for background would be useful. A full Coomassie-blue stained gel (not western blot) is required to demonstrate the direct interaction as with the western blot it cannot be excluded that other proteins bridge the interaction since this is a pull down from lysate not purified proteins. OPTIONAL:Interestingly the surface on Eci1 which binds pex5 is where CoA binds in the active enzyme. Would CoA compete for binding to Pex5? (could add it into the pull down expt?)

    Figure S2: The complex between pex5 and eci1 is solved by cryo EM. Eci1 is hexameric usually 1 but sometimes 2 or 3 pex5s are bound to the complex. The size-exclusion chromatography figure with calculated molecular weight is required to support the stoichiometry. A native gel to show the complex, as well as a denaturing gel (using the complex) to show the individual proteins will be beneficial.

    Figure S9: Would Eci1 compete with Dci1 to bind to Pex5 since they share highly conserved interfaces? If so, why did the deletion of Dci1 impair Eci1 location? Or is this just reduced expression in the dci1 deletion background? (See point 2) This seems counterintuitive/contradictory so please comment.

    OPTIONAL: As the authors acknowledge this work is in vitro. It would have been interesting to examine the role of this interface in vivo by mutating one or more of the residues in Eci 1 identified as being important for the interaction. Granted that mutation can affect the folding of the protein, but the binding region is on the surface so it may not, and this can be readily checked e.g by enzyme activity or limited proteolysis.

    OPTIONAL: Similarly, it would have been interesting to see if mutating the residues of PEX5 involved in the interface affect the import of other cargoes than eci1 or if reciprocal mutations in pex5 and Eci1 e.g switching charges could restore an import defect.

    OPTIONAL If 8 & 9 isn't possible could a co-evolutionary analysis of the interface residues provide further independent evidence for their functional importance? They have looked at conservation of residues in Eci1 but this could be extended to a co-evolution analysis.

    Minor points

    Figure 1C and throughout the manuscript state clearly whether the same confocal settings are used when comparing fluorescence intensity of different images/samples.

    Figure S2B: Please use different colours for PEX5 and Eci1 for clarity.

    Figure 4A: please indicate the PTS1 for the other 5 molecules of Eci1. Are they buried? Or not seen? Please add explanation.

    Figure 4B, C, and D: please colour the circled helix in PEX5 so that it can be more easily seen.

    Please indicate the EBI-mediated interaction in Figure 4C. The relationship between 4C and 4D could be explained better as they are not viewed from the same direction

    Figure S3: As the authors indicated, Pex5 binds with multiple conformations and forms a variable interface with an Eci1 subunit. Does this mean different types of non-canonical interface are possible? Please discuss this.

    Figure 5A and B: they should be labelled as PEX5 TPR domain

    Figure S8 is very helpful in understanding the interface and could be included in Figure 5.

    Significance

    While cargo recognition by Pex5-PTS1 is well understood in molecular detail there are proteins which either lack a PTS1 or have a nonessential PTS1 that still require Pex5 for import into peroxisomes. This study provides a structural view of interaction between Pex5 and its cargo Eci1, a protein that does have a PTS1 but which is not essential for import. It's not the first example of a PEX5-cargo structure to show a non-canonical binding interface and the results are compared to the human pex5-AGT structure. It is an important addition to understanding how so-called PTS context dependent or non1 non2 proteins can be imported. Is this the first structure showing Pex5 bound to an oligomer cargo? Previous work is appropriately cited in the manuscript.

    The study will be of interest to audiences interested in protein-protein interaction and in protein targeting to organelles. This manuscript presents additional knowledge on how an oligomeric PTS1-independent protein can be imported into peroxisomes. The potential of other proteins using the similar importing mechanism can be tested to understand how one receptor can use apparently multiple binding modes to import a wide range of different proteins.

  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

    Peroxisomes are single membrane organelles conserved in all eukaryotes and play important roles in various metabolic reactions, such as beta oxidation of fatty acids. In general, proteins localized in the peroxisomal matrix encode either a C-terminal PTS1 signal or an N-terminal PTS2 signal, and Pex5 acts as a cargo receptor in the PTS1 pathway and Pex7 in the PTS2 pathway, respectively. Previous studies have suggested that some matrix proteins (e.g., Eci1) are transported into the peroxisomal matrix in the PTS1-independent manner, but the mechanism is still unclear. In the present study, Peer et al. determined the Cryo-EM structure of the Pex5-Eci1 complex, which revealed a new interaction site that is distinct from the recognition site of the canonical PTS1 signal, providing important insight into the PTS1-independent, but the Pex5-dependent matrix protein transport. This study by Peer et al. will be of interest to a broad readership in basic cell biology other than peroxisomes.

    The reviewer feels that the manuscript needs to be revised in the following points.

    Major comments

    1. The authors showed that Pex5 binds to Eci1 in a PTS1 signal-independent manner from pull-down experiments in Figure 2, but this result is qualitative. If the authors add quantitative data on the interaction between Pex5 and Eci1 from isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR), this would make this paper more convincing. This could be done in 2 months.
    2. It is not clear to what extent the new interaction sites between Pex5 and Eci1 is important for transport to peroxisomes, as revealed in this study. I suggest, for example, expressing Eci1 with a mutation at a site involved in interaction with Pex5 in yeast and analyzing its effect on peroxisomal localization as additional experiments anew. I believes that this could be done in about 2 months.

    Minor comments

    1. The results of yeast cell imaging in Figures 1, 2 and S1 are all qualitative and not quantitative. Furthermore, there are no descriptions of the experimental reproducibility of the data. I suggest that these points need to be improved.
    2. I feel that information of sample preparation for cryo-EM analysis of the Pex5-Eci1 complex is not enough since it is only described in the methods. I suggest the authors to add the results of gel-filtration chromatography and CBB-stained SDS-PAGE in the manuscript.
    3. The authors discuss the interaction interface between Pex5 and Eci1 in Figures 4 and 5, but the manuscript presented at this stage is difficult at least for me to understand the interaction between them. I recommend the authors to add new figure(s) to show more detailed interaction. Also, I suggest that cryo-EM density map around the interaction region between Pex5 and Eci1 should be presented more detail.

    Significance

    My expertise is in yeast cell biology and structural biology. From this perspective, I think that the strengths of this study are, first, that Pex5-dependent peroxisomal transport of Eci1 in yeast cells occurs independently of PTS1 signal and its paralog Dci1, and that the cryo-EM structure of the Pex5-Eci1 complex reveals a new interaction site other than PTS1 between Pex5 and Eci1. This work is of broad interest not only to peroxisomes, but also to many cell biologists specializing in organelles, and ultimately to structural biologists. On the other hand, the authors' cryo-EM data suggest that 2-3 molecules of Pex5 bind to the Eci1 hexamer. However, it is unclear how the binding of multiple Pex5 molecules to the Eci1 hexamer affects their transport to peroxisomes, and further analysis is needed to elucidate the transport mechanism in more detail.

  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

    Summary:

    Proteins are imported into peroxisomes by mobile receptors such as PEX5. PEX5 recognizes cargo proteins in the cytosol by their peroxisome targeting signal (PTS) and then shuttles them across the peroxisomal membrane into the matrix. While most peroxisomal proteins contain well-characterized signals that bind to PEX5 either directly (PTS1) or through PEX7 (PTS2), some proteins interact with PEX5 independently of these canonical signals. The molecular basis of these unconventional interactions has been poorly understood.

    The manuscript by Peer et al. deals with one such protein called Eci1 in yeast. Eci1 has a PTS1 signal at its C terminus and a putative PTS2 signal at its N terminus, yet the authors show that neither of these signals is required for import of Eci1 into peroxisomes. They also show that import of Eci1 cannot be entirely explained by piggy-backing on its paralog Dci1. Regardless, import of Eci1 depends entirely on PEX5, indicating that Eci1 can bind to PEX5 unconventionally. To identify this additional interface, the authors solve the cryo-EM structure of PEX5 bound to Eci1 (which is a hexamer). Surprisingly, the structure reveals that PEX5 binds to only one of the six Eci1 subunits, and that two distinct interfaces are apparent. One reflects the canonical interaction between the PTS1 signal of Eci1 and the receptor's cognate PTS1-binding TPR domain. The other interface is novel and of potential interest. It involves a region of Eci1 that engages a segment of PEX5 upstream of the TPR domain. This segment has not been previously implicated in binding protein cargo.

    Major issues:

    1. The major issue with the paper is that the novel interface between Eci1 and PEX5 has not been demonstrated to be important for import into peroxisomes. Specifically, mutagenesis of both sides of the interface is required to demonstrate that this interaction mediates import of Eci1 lacking the canonical PTS1 signal (and also in the absence of the paralog Dci1). Such data are indisputably a precondition for publication of this paper. Pull-down experiments should also be performed to demonstrate that the interface is sufficient for interacting with PEX5 in the absence of the PTS1 signal on Eci1.
    2. The paper hinges on the demonstration of a residual interaction between PEX5 and Eci1 lacking its PTS1 signal. However, the pull-down experiment in Figure 3 that allegedly shows this result lacks a critical control for non-specific binding of Eci1 to the nickel beads alone. Also, this experiment does not show a direct interaction between PEX5 and Eci1, since the two proteins are co-expressed in bacteria and then pulled down using an engineered His-tag in PEX5. This experiment should be repeated using PEX5 and Eci1 purified separately and then mixed in vitro. Please show a coomassie-stained SDS-PAGE gel to assess protein purity in addition to the immunoblot, and please show the pull-down in a more conventional way comparing the input and the bound fraction (it is unclear what is meant by soluble and elution fractions).
    3. The presentation of the structure in Figure 4 should be improved. An overview of the complex should be shown first, and then each interface should be pointed out in a different view (and accordingly labeled). It is distracting and not necessary to show all six subunits of Eci1 in different colors. The non-conventional interface should be shown more clearly, with key amino acids numbered and labeled, and the configurations of their side chains highlighted. Please also highlight the salt bridges and hydrogen bonds at this interface that are mentioned in the text but never illustrated.
    4. The data in Figs. S2 and S3 raise doubts about the reported resolution of PEX5 in the cryo-EM structure. Please provide examples of the density map and the fit to the model.
    5. Please provide data for the purification of the complex between PEX5 and Eci1, including a gel-filtration chromatogram and an SDS-PAGE gel of the purified sample used for cryo-EM.
    6. OPTIONAL: The observation that the non-conventional interface between PEX5 with Eci1 corresponds to the site of CoA binding is interesting. This interaction might keep the enzyme inactive while in the cytosol and bound to PEX5, until it would be correctly delivered into peroxisomes and released from the receptor. Alternatively, it could also reflect regulation of Eci1 import by CoA. This idea could easily be tested by pull-down experiments performed with or without CoA, or perhaps by an in vitro Eci1 activity assay in the presence or absence of PEX5. The significance of the paper would be considerably improved if this interaction reflected a mechanism to regulate Eci1 activity or import.

    Minor issues:

    1. The manuscript has many grammatical mistakes which should be addressed. The absence of line numbers precludes us from indicating specific issues.
    2. In general, when referring to a single subunit from the Eci1 hexamer, please use the terms subunit or protomer, and avoid the use of the term monomer which is misleading.
    3. In Fig. 1C, it is unclear whether the experiment was performed in the absence or presence of PEX11. Since the paper hinges on the demonstration of an unconventional interaction between Eci1 and PEX5, perhaps this experiment should be performed in pex11 knockout cells (to enlarge peroxisomes as in Fig. 1B) to show that the residual peroxisomal localization indeed corresponds to the matrix.
    4. In Fig. 6, it would help to show each structure individually and then the overlay.
    5. Fig. S4 should include a scale bar and box size.
    6. Why are phosphorylation sites indicated in Fig. S6?
    7. In Fig. S8, please show the structures of Eci1 bound to PEX5 and to CoA individually, and then the overlay. The figure is very diffucult to understand otherwise.
    8. In Fig. S9, please label the homologous interface residues on Eci1 and Dci1 in individual views, and then show the overlay.

    Significance

    The main finding of the paper is a noncanonical interaction between Eci1 and the peroxisomal import receptor PEX5. This interaction could solve a longstanding mystery about how Eci1 can be targeted to peroxisomes in the absence of its canonical peroxisome targeting signal. Because the authors have not demonstrated that this interaction is sufficient for import of Eci1 in vivo, this key conclusion of the paper remains unconfirmed. If this omission were corrected, the paper would add another example to the growing list of proteins that are imported into peroxisomes by binding unconventionally to PEX5.

    The authors employ an interesting strategy to confirm that Eci1 is correctly imported into the peroxisomal matrix in vivo (and not just recruited to the cytosolic surface of the peroxisomal membrane). This strategy involves enlarging peroxisomes (which normally are diffraction limited) by knocking out a factor required for peroxisome division, allowing the matrix to be resolved from the limiting membrane by light microscopy. Failure to adequately demonstrate import into the matrix had plagued many earlier studies on protein targeting to peroxisomes. The strategy employed in this paper could therefore be useful to other researchers.

    In its current form, the manuscript would be of some interest to the peroxisomal community and perhaps also to researchers studying protein targeting to membrane-bounded organelles. However, if the authors could show that the novel interface between PEX5 and Eci1 functions in part to regulate Eci1 enzymatic activity (or conversely, Eci1 import by CoA), then the paper would be of much broader interest to the fields of metabolism and metabolic regulation.

  5. Version published to 10.1101/2024.06.02.597006v1 on bioRxiv