Exploring the consequences of redirecting an exocytic Rab onto endocytic vesicles

  1. Xia Li
  2. Dongmei Liu
  3. Eric Griffis
  4. Peter Novick

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Abstract

Rab GTPases have been implicated in specifying the direction of vesicle traffic. We test this by redirecting an exocytic Rab onto endocytic vesicles. While defects in both exocytosis and endocytosis are noted, the cells proved surprisingly tolerant of this change.

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  1. Version published to 10.1091/mbc.e23-01-0037
  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

    Reviewer #1 (Evidence, reproducibility and clarity):

    The manuscript from Li et al. describes the authors' attempt to redirect the exocytic Rab Sec4 to endocytic vesicles by fusing the GEF-domain of Sec2 to the CUE domain of the endosomal GEF Vps9, which binds to ubiquitin. The authors show that the localization of the Sec2GEF-GFP-CUE construct is slightly shifted from polarized towards non-polarized sites. Sec2GFP-CUE positive structures acquire Sec4 and Sec4 effectors like exocytic vesicles but are less motile and show delayed plasma membrane fusion. Expression of Sec2GEF-GFP-CUE was enhanced if expressed in a subset of secretory and endocytic mutants and cause delayed Mup1 uptake from the plasma membrane. As Vps9, Sec2GEF-GFP-CUE accumulated on Class E compartments in vps4Δ strains.
    The authors ask here whether vesicular identity is largely predetermined by the correct localization of the specific GEFs of small GTPases and thus localization of the Rab. Although this an interesting hypothesis, the authors observed that endocytic traffic was not reversed by relocating Sec4 to these vesicles. This seems to be due to the strong affinity of the Sec2 GEF-domain for Sec4 but probably also due to the rather weak relocalization via the CUE domain. Thus, only a portion of Sec2 was displaced from its native site. Since the efficiency of this rewiring was not defined, it remains unclear whether the observed mild effects indeed speak against the assumed dominant role of the GEFs and small GTPases in shaping organelle identity or whether they are rather due to an inefficient relocalization.

    Our data demonstrate a dramatic relocalization of Sec2-GEF-GFP-CUE relative to Sec2-GEF-GFP. In the case of Sec2-GEF-GFP or Sec2-GEF-GFP-CUE M419D the cytoplasmic pool is predominant and only 30% of cells exhibit a detectable concentration, while in the case of Sec2-GEF-GFP-CUE 80% of cells show bright puncta and there is little or no detectable cytoplasmic pool (Fig 1A). Clearly the CUE domain can function as a localization domain that relies upon ubiquitin binding. Furthermore, half of the Sec2-GEF-GFP-CUE puncta colocalize with Vps9 (Fig S1). The high cytoplasmic background of Vps9 could mask additional colocalization, therefore we reexamined colocalization in a vps4__D_ _mutant in which the Vps9 cytoplasmic pool is reduced due to increased association with the expanded Class E late endosomes. In this situation we observe about 80% colocalization with Vps9 as well as substantial colocalization with Ypt51 and Vps8 (Fig 2). We now also show significant colocalization with PI(3)P (Fig S3D). Thus, our data demonstrate that addition of the CUE domain does indeed relocalize Sec2GEF to endocytic membranes. The Sec2 GEF activity then leads to the recruitment of Sec4 and Sec4 effectors, including Myo2 which in turn leads to their delivery to polarized sites. We now show by EM that the bright Sec2-GEF-GFP-CUE puncta correlate with clusters of 80 nm vesicles (Fig 5B). Our data argues that these are hybrid compartments carrying both endocytic and exocytic markers. We have restructured our paper to help clarify and emphasize this key point.

    Specific comments:

    1. The authors state decidedly that the recruitment of Vps9 occurs ubiquitin-dependent via the CUE-domain. While the CUE-domain is the only known and a likely localization determinant of Vps9, it was not a strong localization determinant. Apart from being present in some puncta, Vps9 localized strongly to the cytosol (Paulsel et al. 2013, Nagano et al. 2019). Shideler et al. also showed that ubiquitin-binding is not required for Vps9 function in vivo, which indicates that other localizing mechanisms may play a role e. g. by positive feedback of GEF-domain-Rab5 interactions which might be initiated by the other Rab5-GEF Muk1 or as suggested by transport from the Golgi (Nagano et al. 2019). These observations indicate that the CUE-domain is a rather weak recruitment domain, which was not discussed in this manuscript. The localization of the Sec2GEF-GFP-control to the polarized sites in 30% of the cells furthermore suggests that the used Sec2GEF-GFP-CUE retains some native localization via the GEF-domain. Since the relocation efficiency of Sec2GEF-GFP-CUE was not defined, the obtained phenotypic effects allow for only vague conclusions. Although the mild endo- and exocytosis defects as well as the accumulation of Sec2GEF-GFP-CUE at Class E compartments indicate that the CUE-domain indeed conferred some relocation to endosomes, this was not shown for the sec2Δ strain e. g. by looking at colocalizations with endocytic versus exocytic markers and comparing their relative abundance at the Sec2GEF-GFP-CUE-positive structures. While some of the Sec2GEF-GFP-CUE-positive structures colocalized with Mup1 in the Mup1-uptake assay, it would be important to clarify how many endosomal properties are retained and how many exocytic properties are gained by these chimeric vesicles e. g. by looking for the presence of specific phosphoinositides, or Rab5 and Rab5 effectors. A competition between endosomal and the acquired exocytic factors could also be another possible explanation for the immobility of the Sec2GEF-GFP-CUE structures and less efficient recruitment of Sec4 effectors in addition to the proposed lack of PI4P.

    As summarized above, we observed dramatic relocalization of Sec2GEF that was strongly dependent upon the ability of the CUE domain to bind to ubiquitin. We also observed colocalization with Ypt51 and Vps8 as well as transient colocalization with internalized Mup1. We now also show significant colocalization with PI(3)P (Fig S3D). Full length Vps9 is probably subject to additional levels of regulation, perhaps autoinhibitory in nature, however our construct contains only the CUE domain which can clearly function as an efficient localization domain on its own. The high cytoplasmic pool of Vps9 reflects the rapid turnover of its ubiquitin binding sites, since it is efficiently recruited to membranes in vps4__D_ cells. The relocalized Sec2GEF domain was quite effective in recruiting Sec4 as well as most known Sec4 effectors. The recruitment of Myo2 leads to localization to sites of polarized growth. All of our studies were done in a sec2__D _background except for the analysis of dominant growth effects, as now explicitly stated at the beginning of the Results section.

    1. While the colocalization of the Sec2GEF-GFP-CUE-signal with Sec4 indicates that this GEF-construct is generally active, it remains unclear whether the activity of the tagged constructs differ from that of the wild type Sec2 protein. This could be analyzed in vitro via a MANT-GDP GEF-activity assay (Nordmann et al., 2010). Again, it remains unclear how much of the Sec2GEF-Sec4 colocalization represents the retained native localization versus synthetic localization at chimeric endo-exocytic vesicles.

    The structure and nucleotide exchange mechanism of the Sec2 GEF domain have been thoroughly analyzed in prior studies and are well understood. There is no reason to think that the constructs we generated here would alter the exchange activity as the fusions are far removed from the Sec4 binding site and our analysis here confirms that they are active in vivo. We do not feel that there would be much to be gained by doing in vitro exchange assays and it would entail a great deal of work.

    1. The authors mention that tagging with GFP increases the stability of the expressed constructs. However, it remains unclear whether this is also the case for the other tags (NeonGreen, mCherry) used in the other experiments. Are the constructs expressed at similar levels?

    We have compared the levels of the various tagged constructs and they appear to be similar (Fig S5A).

    1. In Figure 5: The incomplete colocalization of Sec2GEF-GFP-CUE with Vps9 is explained by the short-timed accessibility of ubiquitin moieties. Apart from the likely retained native localization or weak CUE-domain-function, this observation could also be due to competition between Vps9 and Sec2GEF-GFP-CUE for the available ubiquitin target structures.

    As previously shown, Vps9 normally displays a prominent cytoplasmic pool. Deletion of Vps4 leads to recruitment of this pool to expanded endosomes through an increase in the lifetime of the ubiquitin binding sites. The high cytoplasmic background in VPS4 cells could obscure some colocalization with Sec2GEF-GFP-CUE and indeed we observe increased colocalization in vps4__D_ _cells in which the cytoplasmic pool of Vps9 has been recruited to endosomes. Expression of Sec2GEF-GFP-CUE does not appear to significantly alter the localization of Vps9.

    Minor remarks:

    1. Fig. 3C do not contain the arrowheads as indicated in the legend, making it harder to interpret.

    These have been added.

    1. The image chosen for Sec2-GFP in Fig. 4B suggests less colocalization between Sec2-GFP and Sec8 than between Sec2GEF-GFP-CUE and Sec8. They rather look next to each other.

    The images initially chosen were not representative. We have replaced them with better images from the same experiment.

    1. Figure 5: While resolution limits are possibly reached regarding endosomes, it might be interesting to check by thin section electron microscopy whether and how class E compartment formation is affected by Sec2GEF-GFP-CUE expression.

    We have now done EM using permanganate fixation of both VPS4 and vps4__D_ cells (Fig 5B and below). In both backgrounds Sec2GEF-GFP-CUE expression leads to the formation of clusters of 80 nm vesicles that appear to correlate with the fluorescent puncta visible by light microscopy. The vps4__D _cells have in addition curved linear membrane structures that represent class E endosomes (see images at end of this file). The class E endosomes appear similar in cells expressing Sec2GEF-GFP-CUE, Sec2-GFP or Sec2. We did not observe any obvious spatial relationship between the class E structures and the vesicle clusters.

    1. Discussion: "Furthermore, delivery of Mup1-GFP to the vacuole was slowed in Sec2GEF-GFP-CUE cells..." - The authors studied "the clearance of Mup1-GFP from the plasma membrane" and not vacuolar delivery. They did not show much vacuolar localization.

    We now include quantitation of Mup1-GFP at both the plasma membrane and vacuole (Fig 6 and Fig S8). This shows a reduced rate of depletion from the plasma membrane and a delayed appearance in the vacuole.

    Literature:
    Nagano, M., Toshima, J. Y., Siekhaus, D. E., & Toshima, J. (2019): Rab5-mediated endosome formation is regulated at the trans-Golgi network. Nature Communications Biology, 2 (1), 1-12.
    Nordmann, M., Cabrera, M., Perz, A., Bröcker, C., Ostrowicz, C., Engelbrecht-Vandré, S., & Ungermann, C. (2010): The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Current Biology, 20(18), 1654-1659.
    Paulsel, A. L., Merz, A. J., & Nickerson, D. P. (2013): Vps9 family protein Muk1 is the second Rab5 guanosine nucleotide exchange factor in budding yeast. Journal of Biological Chemistry, 288 (25), 18162-18171.
    Shideler, T., Nickerson, D. P., Merz, A. J., & Odorizzi, G. (2015): Ubiquitin binding by the CUE domain promotes endosomal localization of the Rab5 GEF Vps9. Molecular Biology of the Cell, 26 (7), 1345-1356.

    Reviewer #1 (Significance):

    • see above
    • has some deficits in interpretation as the Rab relocalization was not complete and thus conclusions are limiting

    Reviewer #2 (Evidence, reproducibility and clarity):

    This paper tries to address a fundamental question in cell biology, namely, what machinery is sufficient to tell a vesicle know where to go and what to do when it gets there. Several groups have shown that localization of some Rab/Ypt GEFs to an orthogonal compartment can lead to redirecting a Rab/Ypt to that membrane, where they can bind their partners abnormally. This story tries to explore what happens next.

    Here, Novick and colleagues took a part of the SEC2 GEF for secretory vesicle SEC4 Rab/Ypt and anchored it to endocytic structures to ask whether that was enough to relocalize those structures and drive inappropriate trafficking events. A challenge and advantage in the study is the fact that not all of the GEF relocalized-and that enables the cells to survive as SEC4p is needed for cell growth and membrane delivery--but this incomplete relocalization complicates phenotypic analysis--some SEC4 is on secretory vesicles and some is relocalized apparently to endocytic structures. Another challenge is that the two compartments both show "polarized" distributions so it is hard to know what compartment the reader is looking at, in a given figure. This makes the story very challenging to digest for a non-yeast expert trying to understand the conclusions.

    The authors show that the CUE domain can serve to partially localize SEC2GEF-GFP-CUE and this function relies on its ability to interact with ubiquitin. The localization is distinct from that of full length Sec2, nonetheless "many structures bearing Sec2GEF-GFP-CUE localize close to the normal sites of cell surface growth despite their abnormal appearance". The authors conclude that SEC4p and its effectors were recruited to these puncta with variable efficiency and the puncta were static; normal secretion was not blocked. This is not really a surprise as some SEC4p is still directed to secretory granules and cells do not show a vesicle accumulation phenotype by EM. Missing seems to be a clear-cut visual assay for exocytosis of secretory granules or endocytic structures despite attempts to include live cell imaging.

    We now show that the bright Sec2GEF-GFP-CUE_ puncta correspond to clusters of 80nm vesicles (Fig 5B). Our FRAP analysis demonstrates that Sec2GEF-GFP-CUE _is able to enter into pre-existing, bleached puncta (Fig 1E). One interpretation is that the vesicle cluster remains static, while individual vesicles enter and exit the cluster.

    The authors showed that SEC2-GFP-CUE structures fail to acquire Sro7 and do not seem to be able to assemble a complex with the tSNARE SEC9. Is this because Sro7 is being retained on the remaining secretory vesicles that also have SEC4 and other effectors that may be recruited to those structures by coordinate recognition?

    We demonstrate that at least half of the Sec2GEF-GFP-CUE puncta colocalize with Vps9 and this becomes even more evident in a vps4__D_ _mutant (Fig 2A). There is also substantial colocalization with the Rab5 homolog Ypt51, the endocytic marker Vps8 and PI(3)P (Fig 2 and Fig S3D). Nearly all of these puncta also colocalize with Sec4 and most of its downstream effectors. Thus, it seems that we have generated a hybrid compartment, as we intended. The surprise is how well the cells can cope with this situation. One possible explanation is offered in the Discussion: In yeast the TGN is thought to play the role of the early endosome and may be the site of Vps9 membrane recruitment. Thus Sec2GEF-GFP-CUE might be initially recruited to the TGN and the hybrid vesicles formed from this compartment might function to bring secretory cargo from the TGN to the cell surface just like normal secretory vesicles, with the caveat that the presence of endocytic machinery is somewhat inhibitory to Sro7 function, slowing fusion.

    There seem to be no issues with data as presented; a diagram of the SEC2-GFP-CUE would help the reader as would use of terms "secretory vesicle" and "endocytic vesicle" and how they were always distinguished rather than "polarized structure" which cannot distinguish these compartments.

    We have tried to be careful in our use of terms. We refer to the Sec2-GFP-CUE puncta using the unbiased terms “structures” or “puncta” until we show EM demonstrating that these puncta represent clusters of 80 nm vesicles.

    CROSS-CONSULTATION COMMENTS
    The two assessments come to the same conclusion--I agree that better definition of the precise phenotypes could be valuable but the limitation of incomplete relocalization will be hard to overcome in the absence of enormous effort.

    Reviewer #2 (Significance):

    This story represents a valiant effort and presents clean data but the impact and significance of the findings are limited due to the difficult phenotypic starting points (SEC4 in two places), and lack powerful exo- or endocytosis assays and better compartment-specific markers.

    The work will be of interest to yeast cell biologists studying the secretory and endocytic pathways. My expertise is mammalian cell biology of the secretory and endocytic pathways.

  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

    This paper tries to address a fundamental question in cell biology, namely, what machinery is sufficient to tell a vesicle know where to go and what to do when it gets there. Several groups have shown that localization of some Rab/Ypt GEFs to an orthogonal compartment can lead to redirecting a Rab/Ypt to that membrane, where they can bind their partners abnormally. This story tries to explore what happens next.

    Here, Novick and colleagues took a part of the SEC2 GEF for secretory vesicle SEC4 Rab/Ypt and anchored it to endocytic structures to ask whether that was enough to relocalize those structures and drive inappropriate trafficking events. A challenge and advantage in the study is the fact that not all of the GEF relocalized-and that enables the cells to survive as SEC4p is needed for cell growth and membrane delivery--but this incomplete relocalization complicates phenotypic analysis--some SEC4 is on secretory vesicles and some is relocalized apparently to endocytic structures. Another challenge is that the two compartments both show "polarized" distributions so it is hard to know what compartment the reader is looking at, in a given figure. This makes the story very challenging to digest for a non-yeast expert trying to understand the conclusions.

    The authors show that the CUE domain can serve to partially localize SEC2GEF-GFP-CUE and this function relies on its ability to interact with ubiquitin. The localization is distinct from that of full length Sec2, nonetheless "many structures bearing Sec2GEF-GFP-CUE localize close to the normal sites of cell surface growth despite their abnormal appearance". The authors conclude that SEC4p and its effectors were recruited to these puncta with variable efficiency and the puncta were static; normal secretion was not blocked. This is not really a surprise as some SEC4p is still directed to secretory granules and cells do not show a vesicle accumulation phenotype by EM. Missing seems to be a clear-cut visual assay for exocytosis of secretory granules or endocytic structures despite attempts to include live cell imaging.

    The authors showed that SEC2-GFP-CUE structures fail to acquire Sro7 and do not seem to be able to assemble a complex with the tSNARE SEC9. Is this because Sro7 is being retained on the remaining secretory vesicles that also have SEC4 and other effectors that may be recruited to those structures by coordinate recognition?

    There seem to be no issues with data as presented; a diagram of the SEC2-GFP-CUE would help the reader as would use of terms "secretory vesicle" and "endocytic vesicle" and how they were always distinguished rather than "polarized structure" which cannot distinguish these compartments.

    Referees cross-commenting

    The two assessments come to the same conclusion--I agree that better definition of the precise phenotypes could be valuable but the limitation of incomplete relocalization will be hard to overcome in the absence of enormous effort.

    Significance

    This story represents a valiant effort and presents clean data but the impact and significance of the findings are limited due to the difficult phenotypic starting points (SEC4 in two places), and lack powerful exo- or endocytosis assays and better compartment-specific markers.

    The work will be of interest to yeast cell biologists studying the secretory and endocytic pathways. My expertise is mammalian cell biology of the secretory and endocytic pathways.

  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 manuscript from Li et al. describes the authors' attempt to redirect the exocytic Rab Sec4 to endocytic vesicles by fusing the GEF-domain of Sec2 to the CUE domain of the endosomal GEF Vps9, which binds to ubiquitin. The authors show that the localization of the Sec2GEF-GFP-CUE construct is slightly shifted from polarized towards non-polarized sites. Sec2GFP-CUE positive structures acquire Sec4 and Sec4 effectors like exocytic vesicles, but are less motile and show delayed plasma membrane fusion. Expression of Sec2GEF-GFP-CUE was enhanced if expressed in a subset of secretory and endocytic mutants and cause delayed Mup1 uptake from the plasma membrane. As Vps9, Sec2GEF-GFP-CUE accumulated on Class E compartments in vps4Δ strains.
    The authors ask here whether vesicular identity is largely predetermined by the correct localization of the specific GEFs of small GTPases and thus localization of the Rab. Although this an interesting hypothesis, the authors observed that endocytic traffic was not reversed by relocating Sec4 to these vesicles. This seems to be due to the strong affinity of the Sec2 GEF-domain for Sec4 but probably also due to the rather weak relocalization via the CUE domain. Thus, only a portion of Sec2 was displaced from its native site. Since the efficiency of this rewiring was not defined, it remains unclear whether the observed mild effects indeed speak against the assumed dominant role of the GEFs and small GTPases in shaping organelle identity or whether they are rather due to an inefficient relocalization.

    Specific comments:

    1. The authors state decidedly that the recruitment of Vps9 occurs ubiquitin-dependent via the CUE-domain. While the CUE-domain is the only known and a likely localization determinant of Vps9, it was not a strong localization determinant. Apart from being present in some puncta, Vps9 localized strongly to the cytosol (Paulsel et al. 2013, Nagano et al. 2019). Shideler et al. also showed that ubiquitin-binding is not required for Vps9 function in vivo, which indicates that other localizing mechanisms may play a role e. g. by positive feedback of GEF-domain-Rab5 interactions which might be initiated by the other Rab5-GEF Muk1 or as suggested by transport from the Golgi (Nagano et al. 2019). These observations indicate that the CUE-domain is a rather weak recruitment domain, which was not discussed in this manuscript. The localization of the Sec2GEF-GFP-control to the polarized sites in 30% of the cells furthermore suggests that the used Sec2GEF-GFP-CUE retains some native localization via the GEF-domain. Since the relocation efficiency of Sec2GEF-GFP-CUE was not defined, the obtained phenotypic effects allow for only vague conclusions. Although the mild endo- and exocytosis defects as well as the accumulation of Sec2GEF-GFP-CUE at Class E compartments indicate that the CUE-domain indeed conferred some relocation to endosomes, this was not shown for the sec2Δ strain e. g. by looking at colocalizations with endocytic versus exocytic markers and comparing their relative abundance at the Sec2GEF-GFP-CUE-positive structures. While some of the Sec2GEF-GFP-CUE-positive structures colocalized with Mup1 in the Mup1-uptake assay, it would be important to clarify how many endosomal properties are retained and how many exocytic properties are gained by these chimeric vesicles e. g. by looking for the presence of specific phosphoinositides, or Rab5 and Rab5 effectors. A competition between endosomal and the acquired exocytic factors could also be another possible explanation for the immobility of the Sec2GEF-GFP-CUE structures and less efficient recruitment of Sec4 effectors in addition to the proposed lack of PI4P.
    2. While the colocalization of the Sec2GEF-GFP-CUE-signal with Sec4 indicates that this GEF-construct is generally active, it remains unclear whether the activity of the tagged constructs differ from that of the wild type Sec2 protein. This could be analyzed in vitro via a MANT-GDP GEF-activity assay (Nordmann et al., 2010). Again, it remains unclear how much of the Sec2GEF-Sec4 colocalization represents the retained native localization versus synthetic localization at chimeric endo-exocytic vesicles.
    3. The authors mention that tagging with GFP increases the stability of the expressed constructs. However, it remains unclear whether this is also the case for the other tags (NeonGreen, mCherry) used in the other experiments. Are the constructs expressed at similar levels?
    4. In Figure 5: The incomplete colocalization of Sec2GEF-GFP-CUE with Vps9 is explained by the short-timed accessibility of ubiquitin moieties. Apart from the likely retained native localization or weak CUE-domain-function, this observation could also be due to competition between Vps9 and Sec2GEF-GFP-CUE for the available ubiquitin target structures.

    Minor remarks:

    1. Fig. 3C do not contain the arrowheads as indicated in the legend, making it harder to interpret.
    2. The image chosen for Sec2-GFP in Fig. 4B suggests less colocalization between Sec2-GFP and Sec8 than between Sec2GEF-GFP-CUE and Sec8. They rather look next to each other.
    3. Figure 5: While resolution limits are possibly reached regarding endosomes, it might be interesting to check by thin section electron microscopy whether and how class E compartment formation is affected by Sec2GEF-GFP-CUE expression.
    4. Discussion: "Furthermore, delivery of Mup1-GFP to the vacuole was slowed in Sec2GEF-GFP-CUE cells..." - The authors studied "the clearance of Mup1-GFP from the plasma membrane" and not vacuolar delivery. They did not show much vacuolar localization.

    Literature:

    Nagano, M., Toshima, J. Y., Siekhaus, D. E., & Toshima, J. (2019): Rab5-mediated endosome formation is regulated at the trans-Golgi network. Nature Communications Biology, 2 (1), 1-12.

    Nordmann, M., Cabrera, M., Perz, A., Bröcker, C., Ostrowicz, C., Engelbrecht-Vandré, S., & Ungermann, C. (2010): The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Current Biology, 20(18), 1654-1659.

    Paulsel, A. L., Merz, A. J., & Nickerson, D. P. (2013): Vps9 family protein Muk1 is the second Rab5 guanosine nucleotide exchange factor in budding yeast. Journal of Biological Chemistry, 288 (25), 18162-18171.

    Shideler, T., Nickerson, D. P., Merz, A. J., & Odorizzi, G. (2015): Ubiquitin binding by the CUE domain promotes endosomal localization of the Rab5 GEF Vps9. Molecular Biology of the Cell, 26 (7), 1345-1356.

    Significance

    • see above
    • has some deficits in interpretation as the Rab relocalization was not complete and thus conclusions are limiting
  5. Version published to 10.1101/2023.02.09.527811 on bioRxiv