Cardiolipin targets a dynamin related protein to the nuclear membrane

  1. Usha Pallabi Kar
  2. Himani Dey
  3. Abdur Rahaman

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Abstract

Dynamins are large cytoplasmic GTPases that are targeted to specific cellular membranes which they remodel via membrane fusion or fission. Although the mechanism of target membrane selection by dynamins has been studied, the molecular basis of conferring specificity to bind specific lipids on the target membranes is not known in any of the family members. Here, we report a mechanism of nuclear membrane recruitment of Drp6 that is involved in nuclear remodeling in Tetrahymena thermophila . Recruitment of Drp6 depends on a domain that binds to cardiolipin-rich bilayers. Consistent with this, the nuclear localization of wildtype Drp6 was inhibited by depleting cardiolipin in the cell. Cardiolipin binding was blocked with a single amino acid substitution (I553M) in the membrane-binding domain of Drp6. Importantly, the I553M substitution was sufficient to block nuclear localization without affecting other properties of Drp6. Consistent with this result, co-expression of wildtype Drp6 was sufficient to rescue the localization defect of I553M variant in Tetrahymena . Inhibition of cardiolipin synthesis or perturbation in Drp6 recruitment to nuclear membrane caused defects in the formation of new macronuclei post-conjugation. Taken together, our results elucidate a molecular basis of target membrane selection by a nuclear dynamin, and establish the importance of a defined membrane-binding domain and its target lipid in facilitating nuclear expansion.

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  1. eLife

    This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on November 19 2020, follows.

    Summary

    The paper reports the involvement of isoleucine 553 in targeting Drp6 to cardiolipin containing nuclear membrane. The data are interesting, but there is no mechanistic understanding of how a single amino acid can target this protein so specifically to cardiolipin enriched membranes.

    Essential Revisions

    The authors are strongly requested to address the issues that were raised in the previous review. The authors state in their rebuttal that they plan to address them in a timely manner. The additional request of one reviewer that should be addressed is to test the involvement of residue 552 and 554 to highlight the significance of isoleucine in position 553 in targeting Drp6 to cardiolipin.

  2. Review Commons

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

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    Reply to the reviewers

    Response to the reviewers’ comments:

    We thank both the reviewer for their critical evaluation and excellent suggestion to improve the manuscript. We are making all the changes suggested by both the reviewers and performing the experiments to address all the concerns specifically from the reviewer #1. Please find below our response to the reviewers’ comments:

    Reviewer #1:

    *This is an interesting study from the Rahaman group that identifies cardiolipin (CL) as a potential binding target for Drp6 recruitment to the nuclear membrane in Tetrahymena (that has a unique nuclear remodeling program). In addition, they identify a residue, I553 in the DTD region, which they claim is a key residue involved in specific CL interactions. While the experiments themselves are technically sound, and are well performed and controlled, I don't find the major conclusion that I553 is involved in direct CL interactions justified or well rationalized. By their own admission (in the discussion), the conservative mutation I553M may perturb local folding and may indirectly affect CL interactions. There is no test of DTD folding with and without the I553M mutation, nor are there other mutations (e.g. I553A and in the vicinity) tested. CD experiments in the absence and presence of CL-containing membranes will likely yield information on the impact of the I553 mutations, while DLS experiments would inform on the hydrodynamic properties (overall 3D fold) of the DTD and the impact of these mutations. CL interactions generally involve a combination of electrostatic and hydrophobic forces. Where do the electrostatic interactions come from? Why would an Isoleucine to Methionine mutation affect the hydrophobic component, even if I553 is the key hydrophobic residue? *

    Response:

    We thank the reviewer for the comments that the experiments are sound, well performed with appropriate controls. While we agree that the exact mechanism of how I553 provides specificity to cardiolipin binding is not addressed in the present manuscript, our study clearly demonstrates that the isoleucine at 553 plays important role in determining cardiolipin specificity and nuclear recruitment. As pointed out by the reviewer, it is possible that changing isoleucine to methionine may affect the local conformation. However, there is no major conformational change in the DTD due to this mutation. This conclusion is based on clear loss of nuclear localization and cardiolipin interaction for the mutant without affecting other properties. The in vitro floatation assay clearly stablish that the effect is directly by inhibiting interaction specifically with cardiolipin containing membrane. It should be further noted that the same domain DTD interacts with other two lipids (PS and PA) and mutant retains interaction with them arguing that conformation of this domain is not significantly changed due to I to M mutation. Consistent with these results I553M mutant could be targeted to the nuclear membrane as a complex with wildtype Drp6 further confirming that I553 could form correct self-assembled structure with wildtype protein required for association with nuclear membrane. This is further substantiated by comparing all the known biochemical properties including GTPase activity, membrane binding via other two lipids, formation of helical spirals and ring structures. Hence it is clear that I553 provides specificity to bind cardiolipin and recruitment to the nuclear membrane. We will further confirm if there is any local conformation change due to the mutation I to M by fluorescence quenching experiments and will be incorporated in the revised manuscript.

    Regarding overall folding of the mutant, this is an excellent suggestion by the reviewer. We are planning to perform CD experiments of the I553M mutant and wildtype proteins to compare if there is any change in overall folding due to mutation. This result would be incorporated in the revised manuscript.

    Reviewer is right to point out that both electrostatic and hydrophobic interactions are important for interaction with cardiolipin. Electrostatic interaction is important for all the phospholipids while interacting with protein and is expected to come from other amino acid residues which are positively charged. Electrostatic interaction may contribute to the affinity of the interaction by providing additional binding energy. But considering its universal nature of interaction with all the phospholipids, it cannot give specificity for a specific lipid and hence would not discriminate among different phospholipids.

    Regarding affecting hydrophobic component, the reviewer is correct that both are strong hydrophobic amino acids and loss of I553M interaction with cardiolipin may not be due to change in hydrophobicity

    To address that the loss of cardiolipin interaction is not specific to methionine and is due to absence of isoleucine, the suggestion from the reviewer to replace I553 with A (alanine) is an excellent one. We are doing the experiments and we anticipate to incorporate these results in our revised manuscript.

    Reviewer #1 (Significance (Required)):

    The addressed phenomenon is restricted to Tetrahymena and may not have far reaching implications. Regardless, the identification of CL as a binding target for Drp6 at the nuclear membrane of this organism is in itself significant. The conclusion that I553 is the key CL binding residue is however not warranted. Additional experiments are needed to dissect how this residue impacts CL interactions and examine whether the observed effect is direct or indirect.*

    Response:

    We thank the reviewer for appreciating the significance of this work. We agree that our data is Tetrahymena specific. However, we believe that the study is relevant for all the proteins whose association with target membranes depend on cardiolipin including many cardiolipin interacting DRPs (such as DRPs involved in biogenesis and maintenance of mitochondria).

    We really appreciate the reviewer for the excellent suggestions. Based on this we are performing the following experiments.

    1. CD experiments to assess overall folding of I553M and Wildtype protein
    2. Fluorescence quenching of Tryptophan (at amino acid position 548) residue in the vicinity of I553 to compare conformation of the mutant with that of wildtype protein.
    3. Evaluation of I553A in nuclear localization and cardiolipin binding. We anticipate these results to further confirm if I553 is the key CL binding residue and if the effect is direct.

    The writing is not clear in some parts and may require a round of language editing. There are no issues with reproducibility.

    Response

    We thank the reviewer for pointing out the language editing. We will edit the language wherever we find it appropriate. We would highly appreciate if reviewer can indicate the portions that need special attention.

    Reviewr #2:

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

    Dynamin is a GTPase superfamily protein involved in membrane fusion and division. This paper focused on Drp6, one of the eight dynamin superfamily proteins of Tetrahymena, and analyzed its nuclear envelope localization mechanism by a combination of in vivo cytogenetical analysis and in vitro biochemical analysis for the various mutant Drp6 proteins. Results showed that a specific amino acid residue (isoleucine at the 553rd) in the membrane binding domain of Drp6 was required for its nuclear membrane localization, but this residue is not required for ER/endosome localization and GTPase activity. Furthermore, in vitro floating analysis using centrifugation indicated that Drp6 specifically bound to the cardiolipin at the 553rd isoleucine residue and this binding was required for Drp6's nuclear membrane localization. Finally, removal of cardiolipin from the conjugating cells using inhibitor treatment showed that cardiolipin was required for the new macronucleus formation (including the expansion of macronuclear envelope) through the function of Drp6. Based on these results, authors concluded that cardiolipin targets Drp6 to the nuclear membrane in Tetrahymena.*

    **Major comments:***

    The experimental data presented in this paper are reasonable and the results are solid, and therefore I think the deduced conclusions are convincing. However, to improve this paper, I have several minor comments to be revised before publication.*

    **Minor comments:***

    In the previous paper, it has been shown that GFP-Drp6 is localized in the inner nuclear membrane of both macronucleus and micronucleus. In this paper, however, this point is not clearly stated and is not shown in the figures --- I could not understand such localization pattern of GFP-Drp6 in Fig. 1C and Fig. 3b and the statements in the text. I suggest adding such statements somewhere in Introduction or Result section. Also, add adequate references to the corresponding statements in the text.*

      • Related to the comment 1, I suggest replacing Fig. 1C (images of fixed cells) with Fig. S1B (images of live cells) because nuclear localization of GFP-Drp6 are much clearer in Fig. S1B (live cell) than Fig. 1C (fixed cell), and because fixation may cause artificial redistribution of the proteins. Please add arrows in those figures to point out the position of micronucleus in those figures if necessary.*
      • Similarly, I suggest replacing images of Fig. 5B (fixed cells) with those of Fig. S3 (live cells).*
      • page 7, line 224: GFP-Nup3 is used as a marker protein of the nuclear pore complex (NPC). However, there is no description of how GFP-Nup3 is obtained or made. Add description how this DNA plasmid was obtained or generated.*
      • Related to the comment 4, "Nup3" is first discovered in Malone et al., Eukaryotic Cells, 2009, but also soon after discovered as the name of "MicNup98B" in Iwamoto et al., Curr Biol, 2009 and used in several papers including Iwamoto et al., Genes Cells, 2010; JCS, 2015; JCS 2017; and more. Because Nup3 is the Tetrahymena paralogs of human Nup98 and the name of "Nup98" is well established to call these homologs in various eukaryotes, I suggest adding the name of "MacNup98B" after the word of "Nup3" for reader's better understanding. I also suggest adding appropriate references to refer to this protein as follows: Add Malone et al. 2009 for "Nup3" and Iwamoto et al., 2009 for "MacNup98B."*
      • page 9, line 295: I wonder if "Fig. 3b" may be a mistake of "Fig. 5C." If so, please correct this.*
      • page 10, the second paragraph (lines 311-322): This paragraph discussed the possible involvement of Drp6 in the nuclear envelope expansion of the post-zygotic nucleus. It may be interesting to point out that large-scale nuclear envelope reorganization including the formation of the redundant nuclear envelope and the type-switching of the NPC (from the MIC-type NPC to the MAC-type one) has been reported at this developmental stage (Iwamoto et al., JCS 2015). For example, the peculiar shaped nuclear envelope with the redundant/overlapping nuclear envelope structure can be seen and the MAC-type NPCs rapidly assembles to the expanding nuclear envelope. It may be interesting to point out that cardiolipin and Drp6 may be involved in these phenomena. But it is too speculative and therefore consider adding such a discussion as an option.*
      • page 13, line 412: Is the word "GFP-drp6-I553M" written in italics intended for the gene for the GFP-drp6-I553M protein? If so, protein may be acceptable here. Make sure there are no problems with italicized characters. Also, check if the lowercase letter "d" in "drp6" is OK because large letters are used in other cases.*
      • page 20, figure 1: I recommend switching the positions of HDyn1 and Drp6 in Figure 1a to keep the order in Figure 1b.*
      • page 21, line 671: Add the word "Tetrahymena" before "Drp 6" to pair with the word "human dynamin 1".*
      • page 23, line 729: Remove "and."*
      • page 23, lines 729 and 731: Unify the expression of "cardiolipin" and "Cardiolipin"*
      • page 23, line 732: Add "or" before "10% Phosphatidylserin."*
      • page 24, Figure 3a: Please mark the position of I553M in the figure if possible. Alternatively, indicate the range of amino acid residues after the words "red" and "green" in the figure legend.* Response:

    We thank the reviewer for the excellent comments that “the experimental data presented in this paper are reasonable and the results are solid, and therefore I think the deduced conclusions are convincing.” We also thank the reviewer for the minor comments which are thorough and very insightful. it will improve the manuscript substantially. We would incorporate all the changes in the revised manuscript.

    Reviewer #2 (Significance (Required)):

    The corresponding author and his colleagues have reported that Tetrahymena Drp6 is localized to the outer nuclear membrane of both macronucleus and micronucleus of Tetrahymena (Elde et al., 2005) and that Drp6 is required for the formation of new macronuclei during nuclear differentiation (Rahaman et al., 2008). Therefore, these parts are not novel.*

    The novelty of this study is as follows:*

    (1) The discovery of a specific amino acid residue (isoleucine at the 553rd) of Drp6 that is required for its nuclear membrane localization.

    (2) the discovery of a lipid molecule, cardiolipin, as a critical partner for Drp6's nuclear membrane targeting.

    (3) Discovery of involvement of cardiolipin in the new macronucleus formation (the expansion of macronuclear envelope) through the function of Drp6.*

    I think their findings are highly novel and will provide new insight into a field of cell biology. Especially, their findings will contribute to understanding how specific proteins targeted to the specific intracellular membranes. In addition, their methods (such as floatation assay) for analyzing the interaction between the protein of interest and lipid/liposomes will become an important tool.*

    Response:

    We are very happy to note that the reviewer has pointed out the significance of the present study. We fully agree with reviewer and appreciate thorough analysis and excellent conclusion from the reviewer.

  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

    Dynamin is a GTPase superfamily protein involved in membrane fusion and division. This paper focused on Drp6, one of the eight dynamin superfamily proteins of Tetrahymena, and analyzed its nuclear envelope localization mechanism by a combination of in vivo cytogenetical analysis and in vitro biochemical analysis for the various mutant Drp6 proteins. Results showed that a specific amino acid residue (isoleucine at the 553rd) in the membrane binding domain of Drp6 was required for its nuclear membrane localization, but this residue is not required for ER/endosome localization and GTPase activity. Furthermore, in vitro floating analysis using centrifugation indicated that Drp6 specifically bound to the cardiolipin at the 553rd isoleucine residue and this binding was required for Drp6's nuclear membrane localization. Finally, removal of cardiolipin from the conjugating cells using inhibitor treatment showed that cardiolipin was required for the new macronucleus formation (including the expansion of macronuclear envelope) through the function of Drp6. Based on these results, authors concluded that cardiolipin targets Drp6 to the nuclear membrane in Tetrahymena.

    Major comments:

    The experimental data presented in this paper are reasonable and the results are solid, and therefore I think the deduced conclusions are convincing. However, to improve this paper, I have several minor comments to be revised before publication.

    Minor comments:

    1. In the previous paper, it has been shown that GFP-Drp6 is localized in the inner nuclear membrane of both macronucleus and micronucleus. In this paper, however, this point is not clearly stated and is not shown in the figures --- I could not understand such localization pattern of GFP-Drp6 in Fig. 1C and Fig. 3b and the statements in the text. I suggest adding such statements somewhere in Introduction or Result section. Also, add adequate references to the corresponding statements in the text.
    2. Related to the comment 1, I suggest replacing Fig. 1C (images of fixed cells) with Fig. S1B (images of live cells) because nuclear localization of GFP-Drp6 are much clearer in Fig. S1B (live cell) than Fig. 1C (fixed cell), and because fixation may cause artificial redistribution of the proteins. Please add arrows in those figures to point out the position of micronucleus in those figures if necessary.
    3. Similarly, I suggest replacing images of Fig. 5B (fixed cells) with those of Fig. S3 (live cells).
    4. page 7, line 224: GFP-Nup3 is used as a marker protein of the nuclear pore complex (NPC). However, there is no description of how GFP-Nup3 is obtained or made. Add description how this DNA plasmid was obtained or generated.
    5. Related to the comment 4, "Nup3" is first discovered in Malone et al., Eukaryotic Cells, 2009, but also soon after discovered as the name of "MicNup98B" in Iwamoto et al., Curr Biol, 2009 and used in several papers including Iwamoto et al., Genes Cells, 2010; JCS, 2015; JCS 2017; and more. Because Nup3 is the Tetrahymena paralogs of human Nup98 and the name of "Nup98" is well established to call these homologs in various eukaryotes, I suggest adding the name of "MacNup98B" after the word of "Nup3" for reader's better understanding. I also suggest adding appropriate references to refer to this protein as follows: Add Malone et al. 2009 for "Nup3" and Iwamoto et al., 2009 for "MacNup98B."
    6. page 9, line 295: I wonder if "Fig. 3b" may be a mistake of "Fig. 5C." If so, please correct this.
    7. page 10, the second paragraph (lines 311-322): This paragraph discussed the possible involvement of Drp6 in the nuclear envelope expansion of the post-zygotic nucleus. It may be interesting to point out that large-scale nuclear envelope reorganization including the formation of the redundant nuclear envelope and the type-switching of the NPC (from the MIC-type NPC to the MAC-type one) has been reported at this developmental stage (Iwamoto et al., JCS 2015). For example, the peculiar shaped nuclear envelope with the redundant/overlapping nuclear envelope structure can be seen and the MAC-type NPCs rapidly assembles to the expanding nuclear envelope. It may be interesting to point out that cardiolipin and Drp6 may be involved in these phenomena. But it is too speculative and therefore consider adding such a discussion as an option.
    8. page 13, line 412: Is the word "GFP-drp6-I553M" written in italics intended for the gene for the GFP-drp6-I553M protein? If so, protein may be acceptable here. Make sure there are no problems with italicized characters. Also, check if the lowercase letter "d" in "drp6" is OK because large letters are used in other cases.
    9. page 20, figure 1: I recommend switching the positions of HDyn1 and Drp6 in Figure 1a to keep the order in Figure 1b.　
    10. page 21, line 671: Add the word "Tetrahymena" before "Drp 6" to pair with the word "human dynamin 1".
    11. page 23, line 729: Remove "and."
    12. page 23, lines 729 and 731: Unify the expression of "cardiolipin" and "Cardiolipin"
    13. page 23, line 732: Add "or" before "10% Phosphatidylserin."
    14. page 24, Figure 3a: Please mark the position of I553M in the figure if possible. Alternatively, indicate the range of amino acid residues after the words "red" and "green" in the figure legend.　

    Significance

    The corresponding author and his colleagues have reported that Tetrahymena Drp6 is localized to the outer nuclear membrane of both macronucleus and micronucleus of Tetrahymena (Elde et al., 2005) and that Drp6 is required for the formation of new macronuclei during nuclear differentiation (Rahaman et al., 2008). Therefore, these parts are not novel.

    The novelty of this study is as follows: (1) The discovery of a specific amino acid residue (isoleucine at the 553rd) of Drp6 that is required for its nuclear membrane localization. (2) the discovery of a lipid molecule, cardiolipin, as a critical partner for Drp6's nuclear membrane targeting. (3) Discovery of involvement of cardiolipin in the new macronucleus formation (the expansion of macronuclear envelope) through the function of Drp6.

    I think their findings are highly novel and will provide new insight into a field of cell biology. Especially, their findings will contribute to understanding how specific proteins targeted to the specific intracellular membranes. In addition, their methods (such as floatation assay) for analyzing the interaction between the protein of interest and lipid/liposomes will become an important tool.

  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

    This is an interesting study from the Rahaman group that identifies cardiolipin (CL) as a potential binding target for Drp6 recruitment to the nuclear membrane in Tetrahymena (that has a unique nuclear remodeling program). In addition, they identify a residue, I553 in the DTD region, which they claim is a key residue involved in specific CL interactions. While the experiments themselves are technically sound, and are well performed and controlled, I don't find the major conclusion that I553 is involved in direct CL interactions justified or well rationalized. By their own admission (in the discussion), the conservative mutation I553M may perturb local folding and may indirectly affect CL interactions. There is no test of DTD folding with and without the I553M mutation, nor are there other mutations (e.g. I553A and in the vicinity) tested. CL interactions generally involve a combination of electrostatic and hydrophobic forces. Where do the electrostatic interactions come from? Why would an Isoleucine to Methionine mutation affect the hydrophobic component, even if I553 is the key hydrophobic residue? Additional experiments are therefore essential to identify the actual residues involved in specific CL interactions. CD experiments in the absence and presence of CL-containing membranes will likely yield information on the impact of the I553 mutations, while DLS experiments would inform on the hydrodynamic properties (overall 3D fold) of the DTD and the impact of these mutations.

    The writing is not clear in some parts and may require a round of language editing. There are no issues with reproducibility.

    Significance

    The addressed phenomenon is restricted to Tetrahymena and may not have far reaching implications. Regardless, the identification of CL as a binding target for Drp6 at the nuclear membrane of this organism is in itself significant. The conclusion that I553 is the key CL binding residue is however not warranted. Additional experiments are needed to dissect how this residue impacts CL interactions and examine whether the observed effect is direct or indirect.

  5. Version published to 10.1101/2020.10.01.322057 on bioRxiv