- Email: [email protected]
Endocytosis: Is There Really a Recycling from Late Endosomes?
Accepted Manuscript Endocytosis: Is there really a Recycling from Late Endosomes? David G. Robinson
PII: DOI: Reference:
S1674-2052(15)00299-3 10.1016/j.molp.2015.07.005 MOLP 157
To appear in: MOLECULAR PLANT Accepted Date: 15 July 2015
Please cite this article as: Robinson D.G. (2015). Endocytosis: Is there really a Recycling from Late Endosomes?. Mol. Plant. doi: 10.1016/j.molp.2015.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
1
ACCEPTED MANUSCRIPT
Endocytosis: Is there really a Recycling from Late Endosomes? David G. Robinson Centre for Organismal Studies, Univ. Heidelberg, D-69120 Heidelberg, Germany
RI PT
[email protected]
Over the last 15 years endocytosis has moved from being a process of only minor importance to plant physiologists to being one of the most exciting research areas in
SC
plant cell biology. These days, nobody doubts the operation of clathrin-mediated endocytosis as a mechanism for the internalization of a range of physiologically
M AN U
important transmembrane protein complexes at the plasma membrane (PM) of plant cells. These include both receptors and transporters. As in animal cells, most of these proteins are constitutively recycled back to the PM from an early endosome (EE). However, some are destined for degradation and proceed further downstream in the endocytic pathway to late endosomes (LE), where they are internalized into the intraluminal vesicles of the LE. Fusion of the LE with the lysosome/vacuole releases
TE D
the vesicles leading to their degradation. The signal which marks PM proteins for degradation has been known in mammalian and yeast cells for quite some time and is polyubiquitination (Mukhopadhyay and Reizman, 2007). In their Spotlight article (Mol Plant, 2015; doi:10.106/j.molp.2015.03.006) Zelazny and Vert highlight the
EP
recent publications from the plant field which also demonstrate a key role for multiple mono-ubiquitination in the endocytosis of the metal transporters IRT1 (Barberon et
AC C
al., 2011), BOR1 (Kasai et al., 2011) as well as lysine63-linked polyubiquitination in vacuolar sorting of the auxin transporter PIN2 (Leitner et al., 2012). They also draw attention to the discovery of a ubiquitin-binding protein (FREE1; also termed FYVE1 by Barberon et al., 2014) which locates to the LE in plant cells and is part of the ESCRT-I (endosomal sorting complex for transport) complex (Gao et al., 2014). This complex sequesters ubiquitinated membrane cargo proteins and internalizes them. Finally, Zelazny and Vert draw attention to the work of Ivanov et al. (2014) in showing an increase in IRT1 degradation and iron deficiency in SNX1 mutants. So far so good, but Zelazny and Vert go on to conclude that a portion of the internalized IRT1 molecules that reach the LE are recycled back to the EE, a process which they
2
ACCEPTED MANUSCRIPT
consider to be mediated by sorting nexin 1 (SNX1), a retromer protein. I question the soundness of this scenario.
My first line of critique centres on the nature of the IRT1 molecules that are present in the LE and their physiological significance. Both Barberon et al. (2011) as well as
RI PT
Ivanov et al. (2014) have documented that around 90% of visualizable IRT1 localizes to the EE (in plants the TGN). Its accumulation at this site is a result of rapid endocytosis at the PM and slow recycling to the PM (Barberon et al., 2014). However, wild type IRT1 is monoubiquitinated at at least two lysines in the cytosolic
SC
loop of this transporter, and gets transported to the vacuole irrespective of the status of iron nutrition. Its detection in the vacuole only being possible when proteolysis is inhibited through concanamycin A treatment (Barberon et al., 2011). Thus it would
M AN U
seem that once internalized, IRT1 molecules get caught up in a cycling loop between the EE and PM before being allowed to proceed further downstream to the LE and then the vacuole. The reason for this is not clear. It could be that some of the incoming IRT1 molecules are de-ubiquinated, and later reubiquitinated. It could also be, as suggested by Barberon et al. (2011), that the degree of ubiquitination is a decisive factor, with single mono-ubiquitination being sufficient for the internalization
TE D
step at the PM but multiple mono-ubiquitinations or lysine63-linked polyubiquitination being required for further downstream trafficking.
EP
Support for the notion that entry into the late endocytic pathway requires that a certain threshold level of ubiquitination be reached on a membrane protein destined for degradation, comes from the studies of Leitner et al. (2012) on PIN2, and Martins
AC C
et al. (2015) on the brassinosteroid hormone receptor BRI1. The former authors compared sorting of a ubiquitination-deficient PIN2-17KR allele, with a constitutively endocytosed PIN2-ubiquitin fusion protein and PIN2-17KR-ubiquitin. They noted that PIN2-17KR-ubiquitin, while still being endocytosed, was no longer efficiently targeted to the vacuole. This suggests that extra ubiquitination is necessary for further downstream transport of the reporter protein to the vacuole. In contrast to IRT1, BRI1 is polyubiquitinated with lysine63-linked chains at residue K866 if not at some of the other 29 lysine residues in BRI1 (Martins et al., 2015). Interestingly, a BRI1 mutant in which 25 of the lysine residues were changed to arginine (essentially a ubiquitindefective mutant), still retained kinase activity, but unlike the wt the BRI125KR mutant
3
ACCEPTED MANUSCRIPT
was mostly present at the PM. Nevertheless, after application of brefeldin A, it was seen to enter BFA-bodies, indicating that the ubiquitin-defective mutant could nevertheless be internalized. However, TIRF imaging of mCitrine-tagged wt BRI1 and the BRI125KR mutant showed that the mutant had a 4-fold longer residency period (28 sec) at the PM. Moreover, in contrast to the wt, BRI125KR-mCitrine was not detectable
RI PT
in the vacuole. These data suggest that while the ubiquitin-defective BRI1 mutant was internalized it failed to gain access to the late endosomal pathway and instead underwent enhanced recycling to the PM from the EE.
SC
Zelazny and Vert draw on the results published by Ivanov et al. (2014), for their claim that SNX1 mediated the recycling of IRT1 from a LE compartment, but care is needed. Ivanov et al. (2014) state that “...unlike IRT1, SNX1 was mainly localized in
M AN U
ARA7-positive compartments“, and that “...29% of the SNX1-positive compartments had no RFP-ARA7 signal showing that part of the SNX1 population is located in different endosomes...“. Thus, from this paper one can only say at best that a portion of the IRT1 colocalizes with SNX1, but with which SNX1-positive compartment remains unclear. This situation is due to the use of the Rab5 GTPase ARA7 as a marker for the LE. As shown in several papers, while ARA7 labels principally the LE,
TE D
it has also been detected at the EE (Stierhof and El Kasmi, 2010; Scheuring et al., 2011; Singh et al., 2014). Since ARA7 is not exclusively a marker for LEs, the conclusion that SNX1 resides at or even recycles IRT1 from LEs is not justified.
EP
Although two papers from the Gaude group (Jaillais et al., 2006; 2008) have claimed that SNX1 locates to a prevacuolar/late endosomal compartment (PVC), the validity of these results has been questioned (Robinson et al., 2012). Moreover, immunogold
AC C
electron microscopy which allows for unequivocal identification of endosomal organelles, has conclusively demonstrated the majority of SNX1 (and SNX2a) at the TGN/EE (Niemes et al., 2010; Stierhof et al., 2013). A function for SNXs at the TGN/EE remains unclear, but SNX1 mutants do not appear to prevent downstream traffic to the vacuole. This has been seen for both IRT1 and PIN2 (Kleine-Vehn et al., 2008; Ivanov et al., 2012). Arabidopsis root cells of SNX1 mutants show reduced levels of both proteins at the plasma membrane and their increased degradation in the vacuole. Similarly, the transient expression of SNX1 and SNX2a mutants in tobacco mesophyll protoplasts is without effect on soluble vacuolar protein transport
4
ACCEPTED MANUSCRIPT
(Niemes et al., 2010). These observations seem to suggest that the SNXs are more involved in recycling to the plasma membrane than in sorting to the vacuole.
Unfortunately, many of the papers where claims for recycling from the LE of plants (morphologically the multivesicular body) have been made, depict the EE (in plants
RI PT
the trans Golgi network, TGN) and LE as static long-lived organelles. Such a situation is not supported by recent research which instead points to the continual production and maturation of a LE from the EE (Scheuring et al., 2011; Singh et al., 2014). Presumably, protein sorting continues during this process, so that depending
SC
on the state of transition one would find different levels of IRT1 (or other PM proteins that are destined for degradation) and different degrees of colocalization between SNX1 and ARA7. If the plant cell goes to such lengths to ensure that only highly
M AN U
ubiquitinated IRT1, BOR1, BRI1, and PIN2 molecules are allowed to enter the late endocytic pathway, it is difficult to understand why such molecules should be retrieved from LEs. What purpose would they fulfill when re-entering an EE?
I feel it is time that the plant community should re-define the LE as a compartment from which there is no recycling. The paper of Jaillais et al. (2008) has been seminal
TE D
in propagating the notion that recycling occurs from the LE. However, their statement the “The PVC, MVB is a crossroads between the secretory and endocytic pathways in Arabidopsis root cells“, is not supported by current opinions which have the two
EP
pathways merging at the TGN/EE (Contento and Bassham, 2012). An underlying reason for this confusion lies I think in an inadequate appreciation of what EE and LE are in mammalian versus plant cells. Recycling of the mannosyl-6-P receptor (MPR)
AC C
from the EE to the TGN in mammalian cells is a consequence of a TGN-based, receptor-mediated sorting mechanism for acid hydrolases which has now become a paradigm for protein sorting in all textbooks. Retromer-mediated recycling of MPRs may still occur as the EE matures into a LE, but there is no evidence for recycling from the LE in mammalian cells (Bulankina et al., 2009).
The problem is that whereas both EE and LE in mammals are multivesiculate structures, in plants only the LE is multivesiculate (hence called a multivesicular body, MVB). Plants also have receptors for segregating lytic enzymes from secretory proteins, known as vacuolar sorting receptors (VSRs). However, it is currently a
5
ACCEPTED MANUSCRIPT
subject of great debate as to where in the plant endomembrane system VSRs connect with and release their ligands. In addition, the localization of plant retromer and the nature of its cargo are also a matter of controversy (for a discussion on these topics see Robinson, 2014). Nevertheless, and in this regard I would like to point to the discovery of the so-called late prevacuolar compartment (LPVC). According to
RI PT
Foresti et al. (2010), this compartment lacks VSRs and is the last endocytic compartment to fuse with the vacuole. Unfortunately, these authors did not provide EM images of their LPVC, but they are most certainly MVB. The budding of vesicles to the outside of plant MVBs has rarely been captured in the EM (the only known
SC
example to my knowledge is Fig. 1 D in Stierhof et al., 2012). From this I infer that if recycling from the late endocytic pathway of plants does occur, it must take place very early in the development of a LE and before completion of the MVB as we
M AN U
recognize it. Therefore I strongly believe that we should define the LE as being an endocytic compartment irreversibly committed for cargo delivery to the lytic vacuole and from which upstream recycling can no longer occur.
References:
TE D
I declare that I have no conflict of interest.
EP
Barberon, M., Zelazny, E., Robert, S., Conejero, G., Curie, C., Friml, J., and Vert, G. (2011) Monoubiquitin-dependent endocytosis of the iron-regulated transporter 1 (IRT1) controls iron uptake in plants. Proc. Natl. Acad. Sci. USA 108:E450-E458.
AC C
Barberon, M., Dubeaux, G., Kolb, C., Isono, E., Zelazny, E., and Vert, G. (2014) Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis. Proc. Natl. Acad. Sci. USA 111:8293–8298.
Bulankina, A.V., Deggerich, A, Wenzel, D., Mutenda, K., Wittmann, J.G., Rudolph, M.G., Burger, K.N.J., and Höning, S. (2009) TIP47 functions in the biogenesis of lipid droplets. J Cell Biol. 185: 641-655 Contento A.L., and Bassham, D.C. (2012) Structure and function of endosomes in plant cells. J. Cell Sci. 125: 3511-3518.
6
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Foresti, O., Gerschlick, D.C., Bottanelli, F., Hummel, E., Hawes, C., and Denecke, J. (2010) A recycling-defective vacuolar sorting receptor reveals an intermediate compartment situated between prevacuoles and vacuoles in tobacco. Plant Cell 22: 3992-4008. Gao, C., Luo, M., Zhao, Q., Kolb, C., Isono, E., Zelazny, E., and Vert, G. (2014) A
RI PT
unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr. Biol. 24: 2556-2563.
Ivanov, R., Brumbarova, T., Blum, A., Jantke, A.M., Finke-Straube, C., and Bauer, P. (2014) SORTING NEXIN 1 is required for modulating the trafficking and
SC
stability of the Arabidopsis IRON-REGULATED TRANSPORTER 1. Plant Cell 26: 1294-1307.
Jaillais, Y., Fobis-Loisy, I., Miege, C., Rollin, C., and Gaude, T. (2006) AtSNX1
M AN U
defines an endosome for auxin-carrier trafficking in Arabidopsis. Nature 443: 106-109 Jaillais, Y., Fobis-Losy, L., Miege, C., and Gaude, T. (2008) Evidence for a sorting endosome in Arabidopsis root cells. Plant J. 53: 237-247
Kasai, K., Takano, J., Miwa, K., Toyoda, A., and Fujiwara, T. (2011) High boroninduced ubiquitinationation regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana. 286: 6175-6183.
TE D
Kleine-Vehn, J., Leitner, J., Zwiewka, M., Sauer, M., Abas, L., Luschnig, C., and Friml, J. (2008) Differential degradation of of PIN2 auxin efflux carrier by retromerdependent vacuolar targeting. Proc. Nat. Acad. Sci (USA) 105: 17812-17817
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Leitner, J., Petrasek, J., Tomanov, K., Retzer, K., Parezova, M.,. Korbei, B., Bachmair, A., Zazimalova, E., and Luschnig, C. (2012) Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation
AC C
of Arabidopsis root growth. Proc. Natl. Acad. Sci. USA 109: 8322-8327 Martins, S., Dohmann, E.M., Cayrel, A., Johnson, A., Fischer, W., Pojer, F., Satiat-Jeunemaitre, B., Jaillais, Y., Chory, J., Geldner, N., and Vert, G. (2015) Internalization and vacuolar targeting of the brassinosteroid hormone receptor BRI1 are regulated by ubiquitination. Nat. Commun. 6: 6151 Mukhopahyay, D., and Riezman, H. (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315: 201-205. Niemes, S., Langhans, M., Viotti, C., Scheuring, D., Yan, M.S.W., Jiang, L., Hillmer, S,. Robinson, D.G., and Pimpl, P. (2010) Retromer recycles vacuolar sorting receptors from the trans-Golgi network. Plant J. 61: 107-121
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Robinson, D.G. (2014) Trafficking of vacuolar sorting receptors: new data and new problems. Plant Physiol. 165: 1417-1423 Robinson, D.G., Pimpl, P., Scheuring, D., Stierhof, Y-D., Sturm, S., and Viotti, C. (2012) Trying to make sense of retromer. Trends Plant Sci. 17: 431-439 Scheuring, D., Viotti, C., Kruger, F., Kunzl, F., Sturm, S., Bubeck, J., Hillmer, S.,
RI PT
Frigerio, L., Robinson, D.G., Pimpl, P., and Schumacher, K. (2011) Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23: 3463-3481.
Singh, M.K., Krüger, F., Beckmann, H., Brumm, S., Vermeer, J.E.M., Munnik, T.,
SC
Mayer, U., Stierhof, Y.D., Grefen, C., Schumacher, K., and Jürgens, G. (2014) Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB-vacuole fusion. Curr. Biol. 24: 1383-13899.
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Stierhof, Y.D., and El Kasmi, F. (2010) Strategies to improve the antigenicity, ultrastructure preservation and visibility of trafficking compartments in Arabidopsis tissue. Eur. J. Cell Biol. 89: 285-297.
Stierhof, Y.D., Viotti, C., Scheuring, D., Sturm, S., and Robinson, D.G. (2013) Sorting nexins 1 and 2a locate mainly to the TGN. Protoplasma 250: 235-240. Zelazny, E., and Vert, G. (2015) Regulation of iron uptake by IRT1: Endocytosis
AC C
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TE D
pulls the trigger. Molecular Plant http:/dx.doi.org/10.1016/j.molp.2015.03.006
PII: DOI: Reference:
S1674-2052(15)00299-3 10.1016/j.molp.2015.07.005 MOLP 157
To appear in: MOLECULAR PLANT Accepted Date: 15 July 2015
Please cite this article as: Robinson D.G. (2015). Endocytosis: Is there really a Recycling from Late Endosomes?. Mol. Plant. doi: 10.1016/j.molp.2015.07.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
1
ACCEPTED MANUSCRIPT
Endocytosis: Is there really a Recycling from Late Endosomes? David G. Robinson Centre for Organismal Studies, Univ. Heidelberg, D-69120 Heidelberg, Germany
RI PT
[email protected]
Over the last 15 years endocytosis has moved from being a process of only minor importance to plant physiologists to being one of the most exciting research areas in
SC
plant cell biology. These days, nobody doubts the operation of clathrin-mediated endocytosis as a mechanism for the internalization of a range of physiologically
M AN U
important transmembrane protein complexes at the plasma membrane (PM) of plant cells. These include both receptors and transporters. As in animal cells, most of these proteins are constitutively recycled back to the PM from an early endosome (EE). However, some are destined for degradation and proceed further downstream in the endocytic pathway to late endosomes (LE), where they are internalized into the intraluminal vesicles of the LE. Fusion of the LE with the lysosome/vacuole releases
TE D
the vesicles leading to their degradation. The signal which marks PM proteins for degradation has been known in mammalian and yeast cells for quite some time and is polyubiquitination (Mukhopadhyay and Reizman, 2007). In their Spotlight article (Mol Plant, 2015; doi:10.106/j.molp.2015.03.006) Zelazny and Vert highlight the
EP
recent publications from the plant field which also demonstrate a key role for multiple mono-ubiquitination in the endocytosis of the metal transporters IRT1 (Barberon et
AC C
al., 2011), BOR1 (Kasai et al., 2011) as well as lysine63-linked polyubiquitination in vacuolar sorting of the auxin transporter PIN2 (Leitner et al., 2012). They also draw attention to the discovery of a ubiquitin-binding protein (FREE1; also termed FYVE1 by Barberon et al., 2014) which locates to the LE in plant cells and is part of the ESCRT-I (endosomal sorting complex for transport) complex (Gao et al., 2014). This complex sequesters ubiquitinated membrane cargo proteins and internalizes them. Finally, Zelazny and Vert draw attention to the work of Ivanov et al. (2014) in showing an increase in IRT1 degradation and iron deficiency in SNX1 mutants. So far so good, but Zelazny and Vert go on to conclude that a portion of the internalized IRT1 molecules that reach the LE are recycled back to the EE, a process which they
2
ACCEPTED MANUSCRIPT
consider to be mediated by sorting nexin 1 (SNX1), a retromer protein. I question the soundness of this scenario.
My first line of critique centres on the nature of the IRT1 molecules that are present in the LE and their physiological significance. Both Barberon et al. (2011) as well as
RI PT
Ivanov et al. (2014) have documented that around 90% of visualizable IRT1 localizes to the EE (in plants the TGN). Its accumulation at this site is a result of rapid endocytosis at the PM and slow recycling to the PM (Barberon et al., 2014). However, wild type IRT1 is monoubiquitinated at at least two lysines in the cytosolic
SC
loop of this transporter, and gets transported to the vacuole irrespective of the status of iron nutrition. Its detection in the vacuole only being possible when proteolysis is inhibited through concanamycin A treatment (Barberon et al., 2011). Thus it would
M AN U
seem that once internalized, IRT1 molecules get caught up in a cycling loop between the EE and PM before being allowed to proceed further downstream to the LE and then the vacuole. The reason for this is not clear. It could be that some of the incoming IRT1 molecules are de-ubiquinated, and later reubiquitinated. It could also be, as suggested by Barberon et al. (2011), that the degree of ubiquitination is a decisive factor, with single mono-ubiquitination being sufficient for the internalization
TE D
step at the PM but multiple mono-ubiquitinations or lysine63-linked polyubiquitination being required for further downstream trafficking.
EP
Support for the notion that entry into the late endocytic pathway requires that a certain threshold level of ubiquitination be reached on a membrane protein destined for degradation, comes from the studies of Leitner et al. (2012) on PIN2, and Martins
AC C
et al. (2015) on the brassinosteroid hormone receptor BRI1. The former authors compared sorting of a ubiquitination-deficient PIN2-17KR allele, with a constitutively endocytosed PIN2-ubiquitin fusion protein and PIN2-17KR-ubiquitin. They noted that PIN2-17KR-ubiquitin, while still being endocytosed, was no longer efficiently targeted to the vacuole. This suggests that extra ubiquitination is necessary for further downstream transport of the reporter protein to the vacuole. In contrast to IRT1, BRI1 is polyubiquitinated with lysine63-linked chains at residue K866 if not at some of the other 29 lysine residues in BRI1 (Martins et al., 2015). Interestingly, a BRI1 mutant in which 25 of the lysine residues were changed to arginine (essentially a ubiquitindefective mutant), still retained kinase activity, but unlike the wt the BRI125KR mutant
3
ACCEPTED MANUSCRIPT
was mostly present at the PM. Nevertheless, after application of brefeldin A, it was seen to enter BFA-bodies, indicating that the ubiquitin-defective mutant could nevertheless be internalized. However, TIRF imaging of mCitrine-tagged wt BRI1 and the BRI125KR mutant showed that the mutant had a 4-fold longer residency period (28 sec) at the PM. Moreover, in contrast to the wt, BRI125KR-mCitrine was not detectable
RI PT
in the vacuole. These data suggest that while the ubiquitin-defective BRI1 mutant was internalized it failed to gain access to the late endosomal pathway and instead underwent enhanced recycling to the PM from the EE.
SC
Zelazny and Vert draw on the results published by Ivanov et al. (2014), for their claim that SNX1 mediated the recycling of IRT1 from a LE compartment, but care is needed. Ivanov et al. (2014) state that “...unlike IRT1, SNX1 was mainly localized in
M AN U
ARA7-positive compartments“, and that “...29% of the SNX1-positive compartments had no RFP-ARA7 signal showing that part of the SNX1 population is located in different endosomes...“. Thus, from this paper one can only say at best that a portion of the IRT1 colocalizes with SNX1, but with which SNX1-positive compartment remains unclear. This situation is due to the use of the Rab5 GTPase ARA7 as a marker for the LE. As shown in several papers, while ARA7 labels principally the LE,
TE D
it has also been detected at the EE (Stierhof and El Kasmi, 2010; Scheuring et al., 2011; Singh et al., 2014). Since ARA7 is not exclusively a marker for LEs, the conclusion that SNX1 resides at or even recycles IRT1 from LEs is not justified.
EP
Although two papers from the Gaude group (Jaillais et al., 2006; 2008) have claimed that SNX1 locates to a prevacuolar/late endosomal compartment (PVC), the validity of these results has been questioned (Robinson et al., 2012). Moreover, immunogold
AC C
electron microscopy which allows for unequivocal identification of endosomal organelles, has conclusively demonstrated the majority of SNX1 (and SNX2a) at the TGN/EE (Niemes et al., 2010; Stierhof et al., 2013). A function for SNXs at the TGN/EE remains unclear, but SNX1 mutants do not appear to prevent downstream traffic to the vacuole. This has been seen for both IRT1 and PIN2 (Kleine-Vehn et al., 2008; Ivanov et al., 2012). Arabidopsis root cells of SNX1 mutants show reduced levels of both proteins at the plasma membrane and their increased degradation in the vacuole. Similarly, the transient expression of SNX1 and SNX2a mutants in tobacco mesophyll protoplasts is without effect on soluble vacuolar protein transport
4
ACCEPTED MANUSCRIPT
(Niemes et al., 2010). These observations seem to suggest that the SNXs are more involved in recycling to the plasma membrane than in sorting to the vacuole.
Unfortunately, many of the papers where claims for recycling from the LE of plants (morphologically the multivesicular body) have been made, depict the EE (in plants
RI PT
the trans Golgi network, TGN) and LE as static long-lived organelles. Such a situation is not supported by recent research which instead points to the continual production and maturation of a LE from the EE (Scheuring et al., 2011; Singh et al., 2014). Presumably, protein sorting continues during this process, so that depending
SC
on the state of transition one would find different levels of IRT1 (or other PM proteins that are destined for degradation) and different degrees of colocalization between SNX1 and ARA7. If the plant cell goes to such lengths to ensure that only highly
M AN U
ubiquitinated IRT1, BOR1, BRI1, and PIN2 molecules are allowed to enter the late endocytic pathway, it is difficult to understand why such molecules should be retrieved from LEs. What purpose would they fulfill when re-entering an EE?
I feel it is time that the plant community should re-define the LE as a compartment from which there is no recycling. The paper of Jaillais et al. (2008) has been seminal
TE D
in propagating the notion that recycling occurs from the LE. However, their statement the “The PVC, MVB is a crossroads between the secretory and endocytic pathways in Arabidopsis root cells“, is not supported by current opinions which have the two
EP
pathways merging at the TGN/EE (Contento and Bassham, 2012). An underlying reason for this confusion lies I think in an inadequate appreciation of what EE and LE are in mammalian versus plant cells. Recycling of the mannosyl-6-P receptor (MPR)
AC C
from the EE to the TGN in mammalian cells is a consequence of a TGN-based, receptor-mediated sorting mechanism for acid hydrolases which has now become a paradigm for protein sorting in all textbooks. Retromer-mediated recycling of MPRs may still occur as the EE matures into a LE, but there is no evidence for recycling from the LE in mammalian cells (Bulankina et al., 2009).
The problem is that whereas both EE and LE in mammals are multivesiculate structures, in plants only the LE is multivesiculate (hence called a multivesicular body, MVB). Plants also have receptors for segregating lytic enzymes from secretory proteins, known as vacuolar sorting receptors (VSRs). However, it is currently a
5
ACCEPTED MANUSCRIPT
subject of great debate as to where in the plant endomembrane system VSRs connect with and release their ligands. In addition, the localization of plant retromer and the nature of its cargo are also a matter of controversy (for a discussion on these topics see Robinson, 2014). Nevertheless, and in this regard I would like to point to the discovery of the so-called late prevacuolar compartment (LPVC). According to
RI PT
Foresti et al. (2010), this compartment lacks VSRs and is the last endocytic compartment to fuse with the vacuole. Unfortunately, these authors did not provide EM images of their LPVC, but they are most certainly MVB. The budding of vesicles to the outside of plant MVBs has rarely been captured in the EM (the only known
SC
example to my knowledge is Fig. 1 D in Stierhof et al., 2012). From this I infer that if recycling from the late endocytic pathway of plants does occur, it must take place very early in the development of a LE and before completion of the MVB as we
M AN U
recognize it. Therefore I strongly believe that we should define the LE as being an endocytic compartment irreversibly committed for cargo delivery to the lytic vacuole and from which upstream recycling can no longer occur.
References:
TE D
I declare that I have no conflict of interest.
EP
Barberon, M., Zelazny, E., Robert, S., Conejero, G., Curie, C., Friml, J., and Vert, G. (2011) Monoubiquitin-dependent endocytosis of the iron-regulated transporter 1 (IRT1) controls iron uptake in plants. Proc. Natl. Acad. Sci. USA 108:E450-E458.
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Barberon, M., Dubeaux, G., Kolb, C., Isono, E., Zelazny, E., and Vert, G. (2014) Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis. Proc. Natl. Acad. Sci. USA 111:8293–8298.
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