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suggest that E-box binding site affinity could account for differences in the expression of core clock genes and clock output genes. First, the top CLK–CYC and CLOCK–BMAL1 binding sites target core clock genes in both Drosophila and mice, respectively [3,6–8]. Second, the core clock gene targets of CLK–CYC and CLOCK–BMAL1 contain dual E-box elements that drive higher levels of transactivation than single E-boxes [8,17], suggesting that they are high-affinity target sites. Third, Drosophila Clk is able to activate both core clock gene expression and circadian oscillator function when expressed in novel locations [18], implying that clock genes have a ‘special status’ that allows them to be activated by CLK–CYC even in ectopic cells. Further studies are required to test whether CLK–CYC and CLOCK–BMAL1 drive target gene expression globally or tissuespecifically depending on target site affinity. To extend the insights from Meireles-Filho et al. further it is also necessary to identify sites bound by CLK–CYC and CLOCK–BMAL1 in specific tissues to accelerate the computational identification of associated binding sites that can be tested for their impact on tissuespecific expression. Identifying factors that bind these nearby sites will enable detailed biochemical studies of factor binding affinity, binding order, and cooperative interactions that promote
tissue-specific transcription and provide a more complete picture of how CLK–CYC and CLOCK–BMAL1 select and activate target gene transcription. References 1. Hardin, P.E., and Panda, S. (2013). Circadian timekeeping and output mechanisms in animals. Curr. Opin. Neurobiol. 23, 724–731. 2. Mohawk, J.A., Green, C.B., and Takahashi, J.S. (2012). Central and peripheral circadian clocks in mammals. Annu. Rev. Neurosci. 35, 445–462. 3. Abruzzi, K.C., Rodriguez, J., Menet, J.S., Desrochers, J., Zadina, A., Luo, W., Tkachev, S., and Rosbash, M. (2011). Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression. Genes Dev. 25, 2374–2386. 4. Ceriani, M.F., Hogenesch, J.B., Yanovsky, M., Panda, S., Straume, M., and Kay, S.A. (2002). Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior. J. Neurosci. 22, 9305–9319. 5. Meireles-Filho, A.C.A., Bardet, A.F., YanezCuna, J.O., Stampfel, G., and Stark, A. (2014). Cis-regulatory requirements for tissue-specific programs of the circadian clock. Curr. Biol. 24, 1–10. 6. Koike, N., Yoo, S.H., Huang, H.C., Kumar, V., Lee, C., Kim, T.K., and Takahashi, J.S. (2012). Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science 338, 349–354. 7. Menet, J.S., Rodriguez, J., Abruzzi, K.C., and Rosbash, M. (2012). Nascent-Seq reveals novel features of mouse circadian transcriptional regulation. eLife 1, e00011. 8. Rey, G., Cesbron, F., Rougemont, J., Reinke, H., Brunner, M., and Naef, F. (2011). Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver. PLoS Biol. 9, e1000595. 9. Panda, S., Antoch, M.P., Miller, B.H., Su, A.I., Schook, A.B., Straume, M., Schultz, P.G., Kay, S.A., Takahashi, J.S., and Hogenesch, J.B. (2002). Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109, 307–320.
Plant Biology: Gatekeepers of the Road to Protein Perdition Targeting membrane proteins for degradation requires the sequential action of ESCRT sub-complexes ESCRT-0 to ESCRT-III. Although this machinery is generally conserved among kingdoms, plants lack the essential ESCRT-0 components. A new report closes this gap by identifying a novel protein family that substitutes for ESCRT-0 function in plants. Michael Sauer1 and Jirı´ Friml2,3 In 1989 the first report appeared about the conserved nature of components of intracellular protein transport between organisms belonging to different kingdoms [1]. Incidentally, it came from the group of
James Rothman, one of this year’s Nobel winners, who, together with Randy Schekman and Thomas Su¨dhof (and certainly many others still waiting for a phone call from Stockholm), pioneered the field of intracellular transport mechanisms. Over the decades, it has become clear that
10. Storch, K.F., Lipan, O., Leykin, I., Viswanathan, N., Davis, F.C., Wong, W.H., and Weitz, C.J. (2002). Extensive and divergent circadian gene expression in liver and heart. Nature 417, 78–83. 11. Vollmers, C., Schmitz, R.J., Nathanson, J., Yeo, G., Ecker, J.R., and Panda, S. (2012). Circadian oscillations of protein-coding and regulatory RNAs in a highly dynamic mammalian liver epigenome. Cell Metab. 16, 833–845. 12. Zaret, K.S., and Carroll, J.S. (2011). Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241. 13. Martinez, G.J., and Rao, A. (2012). Immunology. Cooperative transcription factor complexes in control. Science 338, 891–892. 14. Stefflova, K., Thybert, D., Wilson, M.D., Streeter, I., Aleksic, J., Karagianni, P., Brazma, A., Adams, D.J., Talianidis, I., Marioni, J.C., et al. (2013). Cooperativity and rapid evolution of cobound transcription factors in closely related mammals. Cell 154, 530–540. 15. Hao, H., Glossop, N.R., Lyons, L., Qiu, J., Morrish, B., Cheng, Y., Helfrich-Forster, C., and Hardin, P. (1999). The 69 bp circadian regulatory sequence (CRS) mediates per-like developmental, spatial, and circadian expression and behavioral rescue in Drosophila. J. Neurosci. 19, 987–994. 16. Gertz, J., Savic, D., Varley, K.E., Partridge, E.C., Safi, A., Jain, P., Cooper, G.M., Reddy, T.E., Crawford, G.E., and Myers, R.M. (2013). Distinct properties of cell-type-specific and shared transcription factor binding sites. Mol. Cell 52, 25–36. 17. Paquet, E.R., Rey, G., and Naef, F. (2008). Modeling an evolutionary conserved circadian cis-element. PLoS Comput. Biol. 4, e38. 18. Zhao, J., Kilman, V.L., Keegan, K.P., Peng, Y., Emery, P., Rosbash, M., and Allada, R. (2003). Drosophila clock can generate ectopic circadian clocks. Cell 113, 755–766.
Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, TX 77843-3258, USA. *E-mail: [email protected] http://dx.doi.org/10.1016/j.cub.2013.11.016
much of the molecular machinery is conserved among kingdoms, including not only individual proteins, but also entire complexes. One example is the ESCRT complex, which guides membrane proteins on their way to the lytic organelle (the vacuole in yeast and plants or the lysosome in animals) for degradation. It is composed of four subcomplexes, termed ESCRT-0 to ESCRT-III, that sequentially act on ubiquitinated cargo proteins. The role of ESCRT-0 is the initial recognition and recruitment of ubiquitinated membrane cargo as well as recruitment of ESCRT-I to the membrane. The ESCRT-0 complex is composed of two proteins, Vps27 and HRS in yeast or Hse1 and STAM in
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ESCRT-I ESCRT-II Lytic vacuole ESCRT-III
GAT
TOL
? VHS
Cargo
Ub
PIP
Current Biology
Figure 1. TOL proteins are the functional equivalent of ESCRT-0 in plants. TOLs bind ubiquitinated transmembrane cargo proteins, likely through their VHS/GAT domains. The VHS domain might also play a role for recognition of phosphatidylinositideenriched domains of the plasma membrane. TOLs presumably convey cargo to the ESCRT subcomplexes that will pass the cargo on its way to the vacuole for degradation, but the exact nature of this interaction is currently unresolved.
human. Both complexes contain a characteristic Vps27, HRS and STAM (VHS) domain, recognize ubiquitinated cargo through ubiquitin-interacting motifs (UIMs), bind certain phosphoinositides in the membrane via a FYVE domain and interact with the ESCRT-I complex [2]. The critical and typical VHS domains share structural similarity with the ANTH/ENTH domains found in other important early adaptor proteins, such as epsins required for recruiting proteins into clathrin-coated vesicles [3].
In plants, the ESCRT machinery is generally well conserved, with one notable exception — there are no orthologs of the two ESCRT-0 constituents [4–6]. Therefore, it has been proposed that plants might use an alternative way to load cargo into the ESCRT pathway. Potentially, members of two other protein classes may perform ESCRT-0-like functions: target of Myb (TOM) and Golgi-localized g-ear-containing ARF-binding (GGA) proteins. Both contain the VHS domain at their amino termini and motifs for interaction with ubiquitin,
phosphoinositides and clathrin. Evidence from yeast and animal systems suggests that these proteins may perform similar functions and act in parallel to ESCRT-0 [7]. An extensive phylogenetic analysis of VHS/ANTH/ ENTH proteins in plant genomes revealed no obvious direct TOM1 orthologs, but increased number of proteins containing VHS-GAT (GGA and Tom1 homology) domain similar to GGA proteins [6]. Published recently in Current Biology, Korbei and co-workers [8] have identified a family of nine VHS-GAT domain proteins that they term TOM1-like (TOL). They present an extensive genetic and functional analysis of higher-order loss-offunction mutants, which convincingly demonstrates that TOLs play a crucial function in vacuolar targeting and subsequent degradation of ubiquitinated membrane proteins. Therefore, TOL proteins can be seen as a plant-specific functional substitute for ESCRT-0. It appears that the long sought after molecular player in the first step in ESCRT-mediated degradation in plants is finally found. The capability to regulate the abundance of receptors, transporters or effectors at the cell surface is crucial for cells to respond to both environmental and internal cues. In plants, over the last 15 years, the PIN-FORMED (PIN) family of auxin transporters [9] has emerged as a prominent example of this phenomenon and as a powerful model system for studies on subcellular transport of membrane proteins in general [10]. PIN abundance at the plasma membrane (PM) is controlled by continuous recycling between PM and endosomes, which allows for very rapid and dynamic responses [11] and more ultimately by protein degradation [12]. PINs and another auxin transporter, AUX1, are known to be cargo of the ESCRT complex [13]. Moreover, vacuolar targeting and degradation of PIN2 depends on its ubiquitylation status [14], making PIN2 an excellent model cargo to analyse ESCRT-dependent degradation. Korbei et al. [8] show that higher-order tol mutants are defective in the vacuolar delivery, and therefore degradation, of PIN2 and presumably several other PM proteins. They demonstrate that TOLs bind ubiquitin and ubiquitinated cargo with high specificity (Figure 1). Two
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selected TOLs localize to punctate structures resembling endosomes co-distributing with the trans-Golgi network (TGN) and early endosomes (EE) in close vicinity to the PM. This localization is in accordance with the situation in yeast and animals, where the ESCRT-0 complex recognizes ubiquitinated cargo on early endosomes, recognized by their unique enrichment for certain PIPs. In this respect, it would be interesting to see whether TOLs show specific affinity for certain PIPs. TOLs lack the PIP-interacting FYVE domain found in yeast and animal ESCRT-0, but the VHS domain might functionally substitute for PIP binding activity, as demonstrated in yeast [15]. Inferred from sequence homology, at least some TOL members might also bind clathrin. Again, this would be similar to the behaviour of ESCRT-0, which is known to bind clathrin [16]. Nonetheless, binding of TOLs to clathrin still awaits demonstration. It is currently not clear if there is a preferred location within the endomembrane system for TOL-dependent cargo recognition and recruitment into the later ESCRT subcomplexes. In theory, this recruitment could happen at or close to the PM, but also at the TGN/EE. Indeed, there is a current debate in the plant field about the possibility that the TGN/EE could gradually mature into a multi-vesicular body (MVB)/late endosome (LE) by action of the later ESCRT subcomplexes already at the TGN/EE [17]. However, whether this would necessarily mean that TOL-dependent cargo recognition also occurs at the TGN/EE remains to be seen. In general, it must be admitted that the process of MVB formation and its requirement for degradation of membrane proteins is still a field-in-progress in yeast and animals, and much more so in plants [18]. The same goes for the question of whether cargo ubiquitination is the only way of sorting PM proteins into MVB/LE-dependent degradation. The question of the physiological and developmental importance of the TOLs, however, is not under debate. Among the higher-order mutants, there are several mutant combinations that result in early embryo lethality, indicating the
indispensable nature of these proteins. The viable tol quintuple mutants have very dramatic and somewhat pleiotropic defects. Remarkably, though, many of the phenotypes resemble those found in mutants of components of auxin transport or signalling, such as fused cotyledons, reduced number of lateral roots or problems with tropic responses. Especially the latter phenomenon, reorientation of the root towards the gravity vector, is known to depend strongly on dynamic regulation of PIN2 abundance at the PM. Part of this regulation involves the asymmetric degradation of PIN2 between upper and lower sides of the root [19,20]. Interestingly, TOLs seem to be involved in this process, not only because the tol mutants have gravitropic defects, but also because TOL abundance exhibits a similar spatiotemporally asymmetric pattern. This indicates that TOLs are not just constitutive and static factors required for ESCRT-dependent degradation, but instead undergo dynamic regulation themselves in response to external and internal cues. Such behaviour has so far not been described for ESCRT-0 components in other eukaryotes, and it remains an exciting future question how such a regulation might be achieved. References 1. Wilson, D.W., Wilcox, C.A., Flynn, G.C., Chen, E., Kuang, W.J., Henzel, W.J., Block, M.R., Ullrich, A., and Rothman, J.E. (1989). A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature 339, 355–359. 2. Teis, D., Saksena, S., and Emr, S.D. (2009). SnapShot: the ESCRT machinery. Cell 137, 182–182.e1. 3. Holstein, S.E.H., and Oliviusson, P. (2005). Sequence analysis of Arabidopsis thaliana E/ ANTH-domain-containing proteins: membrane tethers of the clathrin-dependent vesicle budding machinery. Protoplasma 226, 13–21. 4. Winter, V., and Hauser, M.-T. (2006). Exploring the ESCRTing machinery in eukaryotes. Trends Plant Sci. 11, 115–123. 5. Leung, K.F., Dacks, J.B., and Field, M.C. (2008). Evolution of the multivesicular body ESCRT machinery; retention across the eukaryotic lineage. Traffic 9, 1698–1716. 6. De Craene, J.-O., Ripp, R., Lecompte, O., Thompson, J.D., Poch, O., and Friant, S. (2012). Evolutionary analysis of the ENTH/ANTH/VHS protein superfamily reveals a coevolution between membrane trafficking and metabolism. BMC Gen. 13, 297. 7. Wang, T., Liu, N.S., Seet, L.-F., and Hong, W. (2010). The emerging role of VHS domain-containing Tom1, Tom1L1 and Tom1L2 in membrane trafficking. Traffic 11, 1119–1128. 8. Korbei, B., Moulinier-Anzola, J., De-Arajuo, L., Lucyshyn, D., Retzer, K., Khan, M.A., and Luschnig, C. (2013). Arabidopsis TOL proteins act as gatekeepers for vacuolar sorting of PIN2 plasma membrane protein. Curr. Biol. 23, 2500–2505.
ek, J., Mravec, J., Bouchard, R., 9. Petra´s Blakeslee, J.J., Abas, M., Seifertova´, D., niewska, J., Tadele, Z., Kubes , M., Wis ´ , M., et al. (2006). PIN proteins Covanova perform a rate-limiting function in cellular auxin efflux. Science 312, 914–918. 10. Grunewald, W., and Friml, J. (2010). The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells. EMBO J. 29, 2700–2714. 11. Friml, J. (2010). Subcellular trafficking of PIN auxin efflux carriers in auxin transport. Eur. J. Cell Biol. 89, 231–235. 12. Abas, L., Benjamins, R., Malenica, N., niewska, J., Wirniewska, J., Paciorek, T., Wis Moulinier-Anzola, J.C., Sieberer, T., Friml, J., and Luschnig, C. (2006). Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat. Cell Biol. 8, 249–256. 13. Spitzer, C., Reyes, F.C., Buono, R., Sliwinski, M.K., Haas, T.J., and Otegui, M.S. (2009). The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in arabidopsis and are required for plant development. Plant Cell 21, 749–766. ek, J., Tomanov, K., 14. Leitner, J., Petra´s Retzer, K., Parezova´, M., Korbei, B., Bachmair, A., Zaz´ımalova´, E., and Luschnig, C. (2012). Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth. Proc. Natl. Acad. Sci. USA 109, 8322–8327. 15. Demmel, L., Gravert, M., Ercan, E., Habermann, B., Mu¨ller-Reichert, T., Kukhtina, V., Haucke, V., Baust, T., Sohrmann, M., and Kalaidzidis, Y. (2008). The clathrin adaptor Gga2p is a phosphatidylinositol 4-phosphate effector at the Golgi exit. Mol. Biol. Cell 19, 1991–2002. 16. Mayers, J.R., Wang, L., Pramanik, J., Johnson, A., Sarkeshik, A., Wang, Y., Saengsawang, W., Yates, J.R., 3rd, and Audhya, A. (2013). Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane. Proc. Natl. Acad. Sci. USA 110, 11857–11862. 17. Scheuring, D., Viotti, C., Kru¨ger, F., Ku¨nzl, F., Sturm, S., Bubeck, J., Hillmer, S., Frigerio, L., Robinson, D.G., Pimpl, P., et al. (2011). Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23, 3463–3481. 18. Babst, M. (2011). MVB vesicle formation: ESCRT-dependent, ESCRT-independent and everything in between. Curr. Opin. Cell Biol. 23, 452–457. 19. Baster, P., Robert, S., Kleine-Vehn, J., Vanneste, S., Kania, U., Grunewald, W., De Rybel, B., Beeckman, T., and Friml, J. (2013). SCF(TIR1/AFB)-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism. EMBO J. 32, 260–274. 20. Kleine-Vehn, J., Leitner, J., Zwiewka, M., Sauer, M., Abas, L., Luschnig, C., and Friml, J. (2008). Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting. Proc. Natl. Acad. Sci. USA 105, 17812–17817.
1Centro Nacional de Biotecnologı´a, CSIC, 28049 Madrid, Spain. 2Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria. 3Mendel Centre for Plant Genomics and Proteomics, Masaryk University, CEITEC MU, CZ-625 00 Brno, Czech Republic. E-mail: [email protected], jiri.friml@ist. ac.at
http://dx.doi.org/10.1016/j.cub.2013.11.019
suggest that E-box binding site affinity could account for differences in the expression of core clock genes and clock output genes. First, the top CLK–CYC and CLOCK–BMAL1 binding sites target core clock genes in both Drosophila and mice, respectively [3,6–8]. Second, the core clock gene targets of CLK–CYC and CLOCK–BMAL1 contain dual E-box elements that drive higher levels of transactivation than single E-boxes [8,17], suggesting that they are high-affinity target sites. Third, Drosophila Clk is able to activate both core clock gene expression and circadian oscillator function when expressed in novel locations [18], implying that clock genes have a ‘special status’ that allows them to be activated by CLK–CYC even in ectopic cells. Further studies are required to test whether CLK–CYC and CLOCK–BMAL1 drive target gene expression globally or tissuespecifically depending on target site affinity. To extend the insights from Meireles-Filho et al. further it is also necessary to identify sites bound by CLK–CYC and CLOCK–BMAL1 in specific tissues to accelerate the computational identification of associated binding sites that can be tested for their impact on tissuespecific expression. Identifying factors that bind these nearby sites will enable detailed biochemical studies of factor binding affinity, binding order, and cooperative interactions that promote
tissue-specific transcription and provide a more complete picture of how CLK–CYC and CLOCK–BMAL1 select and activate target gene transcription. References 1. Hardin, P.E., and Panda, S. (2013). Circadian timekeeping and output mechanisms in animals. Curr. Opin. Neurobiol. 23, 724–731. 2. Mohawk, J.A., Green, C.B., and Takahashi, J.S. (2012). Central and peripheral circadian clocks in mammals. Annu. Rev. Neurosci. 35, 445–462. 3. Abruzzi, K.C., Rodriguez, J., Menet, J.S., Desrochers, J., Zadina, A., Luo, W., Tkachev, S., and Rosbash, M. (2011). Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression. Genes Dev. 25, 2374–2386. 4. Ceriani, M.F., Hogenesch, J.B., Yanovsky, M., Panda, S., Straume, M., and Kay, S.A. (2002). Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior. J. Neurosci. 22, 9305–9319. 5. Meireles-Filho, A.C.A., Bardet, A.F., YanezCuna, J.O., Stampfel, G., and Stark, A. (2014). Cis-regulatory requirements for tissue-specific programs of the circadian clock. Curr. Biol. 24, 1–10. 6. Koike, N., Yoo, S.H., Huang, H.C., Kumar, V., Lee, C., Kim, T.K., and Takahashi, J.S. (2012). Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science 338, 349–354. 7. Menet, J.S., Rodriguez, J., Abruzzi, K.C., and Rosbash, M. (2012). Nascent-Seq reveals novel features of mouse circadian transcriptional regulation. eLife 1, e00011. 8. Rey, G., Cesbron, F., Rougemont, J., Reinke, H., Brunner, M., and Naef, F. (2011). Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver. PLoS Biol. 9, e1000595. 9. Panda, S., Antoch, M.P., Miller, B.H., Su, A.I., Schook, A.B., Straume, M., Schultz, P.G., Kay, S.A., Takahashi, J.S., and Hogenesch, J.B. (2002). Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109, 307–320.
Plant Biology: Gatekeepers of the Road to Protein Perdition Targeting membrane proteins for degradation requires the sequential action of ESCRT sub-complexes ESCRT-0 to ESCRT-III. Although this machinery is generally conserved among kingdoms, plants lack the essential ESCRT-0 components. A new report closes this gap by identifying a novel protein family that substitutes for ESCRT-0 function in plants. Michael Sauer1 and Jirı´ Friml2,3 In 1989 the first report appeared about the conserved nature of components of intracellular protein transport between organisms belonging to different kingdoms [1]. Incidentally, it came from the group of
James Rothman, one of this year’s Nobel winners, who, together with Randy Schekman and Thomas Su¨dhof (and certainly many others still waiting for a phone call from Stockholm), pioneered the field of intracellular transport mechanisms. Over the decades, it has become clear that
10. Storch, K.F., Lipan, O., Leykin, I., Viswanathan, N., Davis, F.C., Wong, W.H., and Weitz, C.J. (2002). Extensive and divergent circadian gene expression in liver and heart. Nature 417, 78–83. 11. Vollmers, C., Schmitz, R.J., Nathanson, J., Yeo, G., Ecker, J.R., and Panda, S. (2012). Circadian oscillations of protein-coding and regulatory RNAs in a highly dynamic mammalian liver epigenome. Cell Metab. 16, 833–845. 12. Zaret, K.S., and Carroll, J.S. (2011). Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241. 13. Martinez, G.J., and Rao, A. (2012). Immunology. Cooperative transcription factor complexes in control. Science 338, 891–892. 14. Stefflova, K., Thybert, D., Wilson, M.D., Streeter, I., Aleksic, J., Karagianni, P., Brazma, A., Adams, D.J., Talianidis, I., Marioni, J.C., et al. (2013). Cooperativity and rapid evolution of cobound transcription factors in closely related mammals. Cell 154, 530–540. 15. Hao, H., Glossop, N.R., Lyons, L., Qiu, J., Morrish, B., Cheng, Y., Helfrich-Forster, C., and Hardin, P. (1999). The 69 bp circadian regulatory sequence (CRS) mediates per-like developmental, spatial, and circadian expression and behavioral rescue in Drosophila. J. Neurosci. 19, 987–994. 16. Gertz, J., Savic, D., Varley, K.E., Partridge, E.C., Safi, A., Jain, P., Cooper, G.M., Reddy, T.E., Crawford, G.E., and Myers, R.M. (2013). Distinct properties of cell-type-specific and shared transcription factor binding sites. Mol. Cell 52, 25–36. 17. Paquet, E.R., Rey, G., and Naef, F. (2008). Modeling an evolutionary conserved circadian cis-element. PLoS Comput. Biol. 4, e38. 18. Zhao, J., Kilman, V.L., Keegan, K.P., Peng, Y., Emery, P., Rosbash, M., and Allada, R. (2003). Drosophila clock can generate ectopic circadian clocks. Cell 113, 755–766.
Department of Biology and Center for Biological Clocks Research, Texas A&M University, College Station, TX 77843-3258, USA. *E-mail: [email protected] http://dx.doi.org/10.1016/j.cub.2013.11.016
much of the molecular machinery is conserved among kingdoms, including not only individual proteins, but also entire complexes. One example is the ESCRT complex, which guides membrane proteins on their way to the lytic organelle (the vacuole in yeast and plants or the lysosome in animals) for degradation. It is composed of four subcomplexes, termed ESCRT-0 to ESCRT-III, that sequentially act on ubiquitinated cargo proteins. The role of ESCRT-0 is the initial recognition and recruitment of ubiquitinated membrane cargo as well as recruitment of ESCRT-I to the membrane. The ESCRT-0 complex is composed of two proteins, Vps27 and HRS in yeast or Hse1 and STAM in
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ESCRT-I ESCRT-II Lytic vacuole ESCRT-III
GAT
TOL
? VHS
Cargo
Ub
PIP
Current Biology
Figure 1. TOL proteins are the functional equivalent of ESCRT-0 in plants. TOLs bind ubiquitinated transmembrane cargo proteins, likely through their VHS/GAT domains. The VHS domain might also play a role for recognition of phosphatidylinositideenriched domains of the plasma membrane. TOLs presumably convey cargo to the ESCRT subcomplexes that will pass the cargo on its way to the vacuole for degradation, but the exact nature of this interaction is currently unresolved.
human. Both complexes contain a characteristic Vps27, HRS and STAM (VHS) domain, recognize ubiquitinated cargo through ubiquitin-interacting motifs (UIMs), bind certain phosphoinositides in the membrane via a FYVE domain and interact with the ESCRT-I complex [2]. The critical and typical VHS domains share structural similarity with the ANTH/ENTH domains found in other important early adaptor proteins, such as epsins required for recruiting proteins into clathrin-coated vesicles [3].
In plants, the ESCRT machinery is generally well conserved, with one notable exception — there are no orthologs of the two ESCRT-0 constituents [4–6]. Therefore, it has been proposed that plants might use an alternative way to load cargo into the ESCRT pathway. Potentially, members of two other protein classes may perform ESCRT-0-like functions: target of Myb (TOM) and Golgi-localized g-ear-containing ARF-binding (GGA) proteins. Both contain the VHS domain at their amino termini and motifs for interaction with ubiquitin,
phosphoinositides and clathrin. Evidence from yeast and animal systems suggests that these proteins may perform similar functions and act in parallel to ESCRT-0 [7]. An extensive phylogenetic analysis of VHS/ANTH/ ENTH proteins in plant genomes revealed no obvious direct TOM1 orthologs, but increased number of proteins containing VHS-GAT (GGA and Tom1 homology) domain similar to GGA proteins [6]. Published recently in Current Biology, Korbei and co-workers [8] have identified a family of nine VHS-GAT domain proteins that they term TOM1-like (TOL). They present an extensive genetic and functional analysis of higher-order loss-offunction mutants, which convincingly demonstrates that TOLs play a crucial function in vacuolar targeting and subsequent degradation of ubiquitinated membrane proteins. Therefore, TOL proteins can be seen as a plant-specific functional substitute for ESCRT-0. It appears that the long sought after molecular player in the first step in ESCRT-mediated degradation in plants is finally found. The capability to regulate the abundance of receptors, transporters or effectors at the cell surface is crucial for cells to respond to both environmental and internal cues. In plants, over the last 15 years, the PIN-FORMED (PIN) family of auxin transporters [9] has emerged as a prominent example of this phenomenon and as a powerful model system for studies on subcellular transport of membrane proteins in general [10]. PIN abundance at the plasma membrane (PM) is controlled by continuous recycling between PM and endosomes, which allows for very rapid and dynamic responses [11] and more ultimately by protein degradation [12]. PINs and another auxin transporter, AUX1, are known to be cargo of the ESCRT complex [13]. Moreover, vacuolar targeting and degradation of PIN2 depends on its ubiquitylation status [14], making PIN2 an excellent model cargo to analyse ESCRT-dependent degradation. Korbei et al. [8] show that higher-order tol mutants are defective in the vacuolar delivery, and therefore degradation, of PIN2 and presumably several other PM proteins. They demonstrate that TOLs bind ubiquitin and ubiquitinated cargo with high specificity (Figure 1). Two
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selected TOLs localize to punctate structures resembling endosomes co-distributing with the trans-Golgi network (TGN) and early endosomes (EE) in close vicinity to the PM. This localization is in accordance with the situation in yeast and animals, where the ESCRT-0 complex recognizes ubiquitinated cargo on early endosomes, recognized by their unique enrichment for certain PIPs. In this respect, it would be interesting to see whether TOLs show specific affinity for certain PIPs. TOLs lack the PIP-interacting FYVE domain found in yeast and animal ESCRT-0, but the VHS domain might functionally substitute for PIP binding activity, as demonstrated in yeast [15]. Inferred from sequence homology, at least some TOL members might also bind clathrin. Again, this would be similar to the behaviour of ESCRT-0, which is known to bind clathrin [16]. Nonetheless, binding of TOLs to clathrin still awaits demonstration. It is currently not clear if there is a preferred location within the endomembrane system for TOL-dependent cargo recognition and recruitment into the later ESCRT subcomplexes. In theory, this recruitment could happen at or close to the PM, but also at the TGN/EE. Indeed, there is a current debate in the plant field about the possibility that the TGN/EE could gradually mature into a multi-vesicular body (MVB)/late endosome (LE) by action of the later ESCRT subcomplexes already at the TGN/EE [17]. However, whether this would necessarily mean that TOL-dependent cargo recognition also occurs at the TGN/EE remains to be seen. In general, it must be admitted that the process of MVB formation and its requirement for degradation of membrane proteins is still a field-in-progress in yeast and animals, and much more so in plants [18]. The same goes for the question of whether cargo ubiquitination is the only way of sorting PM proteins into MVB/LE-dependent degradation. The question of the physiological and developmental importance of the TOLs, however, is not under debate. Among the higher-order mutants, there are several mutant combinations that result in early embryo lethality, indicating the
indispensable nature of these proteins. The viable tol quintuple mutants have very dramatic and somewhat pleiotropic defects. Remarkably, though, many of the phenotypes resemble those found in mutants of components of auxin transport or signalling, such as fused cotyledons, reduced number of lateral roots or problems with tropic responses. Especially the latter phenomenon, reorientation of the root towards the gravity vector, is known to depend strongly on dynamic regulation of PIN2 abundance at the PM. Part of this regulation involves the asymmetric degradation of PIN2 between upper and lower sides of the root [19,20]. Interestingly, TOLs seem to be involved in this process, not only because the tol mutants have gravitropic defects, but also because TOL abundance exhibits a similar spatiotemporally asymmetric pattern. This indicates that TOLs are not just constitutive and static factors required for ESCRT-dependent degradation, but instead undergo dynamic regulation themselves in response to external and internal cues. Such behaviour has so far not been described for ESCRT-0 components in other eukaryotes, and it remains an exciting future question how such a regulation might be achieved. References 1. Wilson, D.W., Wilcox, C.A., Flynn, G.C., Chen, E., Kuang, W.J., Henzel, W.J., Block, M.R., Ullrich, A., and Rothman, J.E. (1989). A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature 339, 355–359. 2. Teis, D., Saksena, S., and Emr, S.D. (2009). SnapShot: the ESCRT machinery. Cell 137, 182–182.e1. 3. Holstein, S.E.H., and Oliviusson, P. (2005). Sequence analysis of Arabidopsis thaliana E/ ANTH-domain-containing proteins: membrane tethers of the clathrin-dependent vesicle budding machinery. Protoplasma 226, 13–21. 4. Winter, V., and Hauser, M.-T. (2006). Exploring the ESCRTing machinery in eukaryotes. Trends Plant Sci. 11, 115–123. 5. Leung, K.F., Dacks, J.B., and Field, M.C. (2008). Evolution of the multivesicular body ESCRT machinery; retention across the eukaryotic lineage. Traffic 9, 1698–1716. 6. De Craene, J.-O., Ripp, R., Lecompte, O., Thompson, J.D., Poch, O., and Friant, S. (2012). Evolutionary analysis of the ENTH/ANTH/VHS protein superfamily reveals a coevolution between membrane trafficking and metabolism. BMC Gen. 13, 297. 7. Wang, T., Liu, N.S., Seet, L.-F., and Hong, W. (2010). The emerging role of VHS domain-containing Tom1, Tom1L1 and Tom1L2 in membrane trafficking. Traffic 11, 1119–1128. 8. Korbei, B., Moulinier-Anzola, J., De-Arajuo, L., Lucyshyn, D., Retzer, K., Khan, M.A., and Luschnig, C. (2013). Arabidopsis TOL proteins act as gatekeepers for vacuolar sorting of PIN2 plasma membrane protein. Curr. Biol. 23, 2500–2505.
ek, J., Mravec, J., Bouchard, R., 9. Petra´s Blakeslee, J.J., Abas, M., Seifertova´, D., niewska, J., Tadele, Z., Kubes , M., Wis ´ , M., et al. (2006). PIN proteins Covanova perform a rate-limiting function in cellular auxin efflux. Science 312, 914–918. 10. Grunewald, W., and Friml, J. (2010). The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells. EMBO J. 29, 2700–2714. 11. Friml, J. (2010). Subcellular trafficking of PIN auxin efflux carriers in auxin transport. Eur. J. Cell Biol. 89, 231–235. 12. Abas, L., Benjamins, R., Malenica, N., niewska, J., Wirniewska, J., Paciorek, T., Wis Moulinier-Anzola, J.C., Sieberer, T., Friml, J., and Luschnig, C. (2006). Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat. Cell Biol. 8, 249–256. 13. Spitzer, C., Reyes, F.C., Buono, R., Sliwinski, M.K., Haas, T.J., and Otegui, M.S. (2009). The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in arabidopsis and are required for plant development. Plant Cell 21, 749–766. ek, J., Tomanov, K., 14. Leitner, J., Petra´s Retzer, K., Parezova´, M., Korbei, B., Bachmair, A., Zaz´ımalova´, E., and Luschnig, C. (2012). Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth. Proc. Natl. Acad. Sci. USA 109, 8322–8327. 15. Demmel, L., Gravert, M., Ercan, E., Habermann, B., Mu¨ller-Reichert, T., Kukhtina, V., Haucke, V., Baust, T., Sohrmann, M., and Kalaidzidis, Y. (2008). The clathrin adaptor Gga2p is a phosphatidylinositol 4-phosphate effector at the Golgi exit. Mol. Biol. Cell 19, 1991–2002. 16. Mayers, J.R., Wang, L., Pramanik, J., Johnson, A., Sarkeshik, A., Wang, Y., Saengsawang, W., Yates, J.R., 3rd, and Audhya, A. (2013). Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane. Proc. Natl. Acad. Sci. USA 110, 11857–11862. 17. Scheuring, D., Viotti, C., Kru¨ger, F., Ku¨nzl, F., Sturm, S., Bubeck, J., Hillmer, S., Frigerio, L., Robinson, D.G., Pimpl, P., et al. (2011). Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23, 3463–3481. 18. Babst, M. (2011). MVB vesicle formation: ESCRT-dependent, ESCRT-independent and everything in between. Curr. Opin. Cell Biol. 23, 452–457. 19. Baster, P., Robert, S., Kleine-Vehn, J., Vanneste, S., Kania, U., Grunewald, W., De Rybel, B., Beeckman, T., and Friml, J. (2013). SCF(TIR1/AFB)-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism. EMBO J. 32, 260–274. 20. Kleine-Vehn, J., Leitner, J., Zwiewka, M., Sauer, M., Abas, L., Luschnig, C., and Friml, J. (2008). Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting. Proc. Natl. Acad. Sci. USA 105, 17812–17817.
1Centro Nacional de Biotecnologı´a, CSIC, 28049 Madrid, Spain. 2Institute of Science and Technology Austria, 3400 Klosterneuburg, Austria. 3Mendel Centre for Plant Genomics and Proteomics, Masaryk University, CEITEC MU, CZ-625 00 Brno, Czech Republic. E-mail: [email protected], jiri.friml@ist. ac.at
http://dx.doi.org/10.1016/j.cub.2013.11.019
