DOI 10.1515/hsz-2013-0258 Biol. Chem. 2014; 395(3): 327–333
Minireview Johannes Numrich and Christian Ungermann*
Endocytic Rabs in membrane trafficking and signaling Abstract: The endolysosomal system controls the trafficking of proteins between the plasma membrane and the degradative environment of the lysosome. The early endosomal Rab5 and the late endosomal Rab7 GTPases have a key role in the transport along the endocytic pathway by recruiting tethering factors such as the hexameric CORVET and HOPS complexes that promote membrane fusion. Both Rabs are also involved in signaling at endosomal membranes and linked to amino acid sensing and autophagy, indicating that their role in trafficking may be connected to signal transduction and adaptation during cell stress. Here, we will summarize the current knowledge on the role of both Rab GTPases on both processes and discuss the possible crosstalk between them. Keywords: endosome; Rab5; Rab7; signaling; trafficking; vacuole; Vps21; Ypt7. *Corresponding author: Christian Ungermann, Department of Biology/Chemistry, Biochemistry Section, University of Osnabrück, Barbarastrasse 13, D-49076 Osnabrück, Germany, e-mail: [email protected] Johannes Numrich: Department of Biology/Chemistry, Biochemistry Section, University of Osnabrück, D-49076 Osnabrück, Germany
Introduction Eukaryotic cells have a highly dynamic endomembrane system, in which organelles are interconnected by vesicular carriers that transport both lipids and proteins (Bonifacino and Glick, 2004). Vesicles form by fission at one organelle and may use the cytoskeleton for long-distance trafficking, delivering their content by subsequent fusion with the target membrane. Whereas fission depends on coat proteins, fusion requires the interplay of a conserved machinery that consists of Rab GTPases, tethering factors and SNAREs. Rab GTPases are considered as organelle-specific markers, which recruit tethering factors to the membrane surface. Tethers also bind to incoming vesicles, via either a vesicular Rab GTPase, SNAREs or lipid, and thus, mediate the first contact of membranes. Once the two membranes are close enough, SNAREs on the vesicle and target membranes zipper
together, and provide the energy for fusion of the proximal lipid bilayers. The identification of the basic mechanism of these processes were acknowledged by granting this years the Nobel prize in Medicine or Physiology to Randy Scheckman, James Rothman and Thomas Südhof.
Rab function Rab GTPases are the key regulators of fusion events that occur in the endomembrane system (Barr, 2013; Barr and Lambright, 2010; Hutagalung and Novick, 2011; Itzen and Goody, 2011). The recruitment of a specific Rab determines the identity of each organelle. The activity of a Rab is provided by its cycle between the inactive GDP- and the active GTP-loaded form, which is then able to interact with effectors such as tethers. Rabs are poor GTPases, and should rather be considered as molecular switches that require co-factors for cycling between their different states (Bos et al., 2007; Itzen and Goody, 2011). Within the cytosol, the GDP-bound Rab is kept soluble by a GDP dissociation inhibitor (GDI), which interacts with the C-terminal prenyl anchor and shields the nucleotide-binding pocket of the N-terminal GTPase domain. To be recruited onto membranes, the GDI has to be released. This process may require a GDI displacement factor (GDF), although its general necessity is unclear. Instead, the activator of the Rab, the guanine nucleotide exchange factor (GEF), may be sufficient to displace both GDI and promote nucleotide exchange (Itzen and Goody, 2011). In the GTP-loaded state, Rabs are stably associated to a membrane via their C-terminal prenyl anchor, and can interact with their respective effectors. As a result of their low hydrolysis rate, Rabs depend on GTPase activating proteins (GAPs), which complement the active site and promotes GTP hydrolysis. The Rab-GDP may then be either extracted from the membrane by GDI or undergo activation again (see above references). Within this review, we will focus exclusively on the yeast Rab GTPases Vps21 and Ypt7 and their metazoan homologs Rab5 and Rab7, which are key regulators of endosomal biogenesis, maturation and fusion (Figure 1, bottom part). We will mostly concentrate on observations made in yeast, and will refer to higher eukaryotes in selected examples.
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328 J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling
Figure 1 Endosomal maturation is linked to TORC1 activation. Trafficking from early endosomes to late endosomes and lysosomes is dependent on Rab5 and Rab7 function. These Rabs recruit the tethering complexes CORVET and HOPS to initiate vesicle fusion and the Vps34 Kinase, which produces phosphoinositide-3-phosphates (PI-3-P) at endosomes. PI-3-P in turn is needed for effector recruitment. Loss of Rab5 or Rab7 function leads to altered PI-3-P levels at endosomes, resulting, on the one hand, in defective endosomal maturation, and on the other hand, in reduced TORC1 activity. If PI-3-P levels directly modulate TORC1 activity via recruitment of regulators of TORC1, or indirectly via influencing endosomal maturation and thereby flux of amino acids, remains to be shown. PM, plasma membrane; EV, endocytic vesicle; LE, late endosome; L/V, lysosome/vacuole.
Rab interactions and regulation within the endocytic pathway
Regulation of the Rab5 GTPase Vps21 by GEFs and GAPs
Early endosomes receive cargoes like cell surface receptors or vacuolar hydrolases via vesicular carriers from the trans Golgi network (TGN) or from the plasma membrane. In yeast, the Rab5-like GTPase Vps21 and its effectors, the CORVET tethering complex and the EEA1 homolog Vac1, mediate the fusion of these carriers with the early endosome (Peterson et al., 1999; Tall et al., 1999; Peplowska et al., 2007; Cabrera et al., 2013; Epp and Ungermann, 2013). Our data suggest that Vac1-mediated fusion is primarily required to fuse endocytic vesicles with endosomes (Cabrera et al., 2013). CORVET, as a heterohexamer, has two subunits that bind to Vps21-GTP, and likely acts in homotypic fusion of endosomes (Peplowska et al., 2007; Balderhaar et al., 2013). Early endosomes subsequently mature into Rab7/Ypt7-positive late endosomes, also called MVBs (multivesicular bodies), which finally fuse with vacuoles (Balderhaar and Ungermann, 2013). During this maturation process, ubiquitinated cell surface receptors are sorted into intraluminal vesicles with the help of the ESCRT protein complexes, whereas non-ubiquitinated receptors are recycled back to the plasma membrane. The latter process depends on the Retromer complex (Seaman et al., 2013). Once the surface of a MVB is cleared from all ubiquitinated receptors, the recruitment and activation of the Ypt7 protein and its effector, the hexameric HOPS complex seems to occur downstream and promote tethering and fusion of mature MVBs with vacuoles (see Figure 1).
The Rab5 homolog Vps21 regulates homotypic fusion of endocytic vesicles and heterotypic fusion events of endocytic or TGN-derived vesicles with the early endosome. Beside Vps21, two additional Rab5 homologs named Ypt52 and Ypt53 act at endosomal membranes (Singer-Kruger et al., 1994). Both have redundant roles with Vps21 (Nickerson et al., 2012; Cabrera et al., 2013), and Ypt53 is mainly expressed during environmental stress (Liu et al., 2011; Nickerson et al., 2012). The most important Rab5 homolog Vps21 is activated by its GEF Vps9, which triggers the exchange of GDP to GTP (Hama et al., 1999). Recent studies identified Muk1 as a second protein with GEF activity towards Vps21 (Cabrera et al., 2013; Paulsel et al., 2013). Muk1 is not necessary for proper sorting of the endosomal cargo protein carboxypeptidase S (Cps1) into intraluminal vesicles of MVBs, indicating that it might not be critical for the activation of Vps21 in vivo. In contrast, a vps9 deletion mutant has the same defect in the missorting of Cps1 as a vps21 mutant (Cabrera et al., 2013). However, Muk1 seems to replace Vps9 partially (Cabrera et al., 2013; Paulsel et al., 2013), and Vps21 is only completely mislocalized in the absence of both GEFs (Cabrera and Ungermann, 2013). Thus, two Rab5-GEFs with some overlapping function are required in yeast, similar to the situation in metazoan cells, where multiple Rab5-GEFs have been identified (Carney et al., 2006). Vps21 is subsequently inactivated by the GAP protein Msb3 (Lachmann et al., 2012; Nickerson et al.,
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J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling 329
2012). Interestingly, Msb3 has been identified as the GAP of the secretory Rab Sec4 (Gao et al., 2003), and requires the heterohexameric BLOC-1 complex for its targeting to endosomal membranes (John Peter et al., 2013). Furthermore, Msb3 has also activity for the Rab7-like Ypt7 protein of the vacuole (Lachmann et al., 2012; Nickerson et al., 2012), suggesting that Msb3 controls the rate of endocytic transport by inactivation of multiple Rabs.
Vps21 effectors Once activated, Vps21 recruits its effectors, the EEA1-like Vac1 and the hexameric CORVET complex, to the endosomal surface. Vac1 belongs to the family of so-called coiled coil tethers, seems to function upstream of CORVET (Cabrera et al., 2013), and is needed for the fusion of endocytic vesicles (Peterson et al., 1999; Tall et al., 1999), whereas the CORVET complex is likely required for homotypic fusion of early endosomes (Balderhaar et al., 2013). The hexameric CORVET complex consists of the four class C subunits (Vps11, Vps16, Vps18 and the Sec1/Munc18 homolog Vps33) and two additional subunits named Vps3 and Vps8 (Peplowska et al., 2007). All subunits except Vps33 have structural homology, and contain a predicted N-terminal b-propeller and a C-terminal α-solenoid domain (Nickerson et al., 2009). The subunits Vps3 and Vps8 interact with the Rab GTPase Vps21 directly (Horazdovsky et al., 1996; Markgraf et al., 2009; Pawelec et al., 2010; Plemel et al., 2011; Epp and Ungermann, 2013). Whether both or only one subunit is needed for the recruitment of the CORVET to endosomes remains unclear (Epp and Ungermann, 2013). Once recruited to the membrane, CORVET promotes tethering of endosomal membranes, and thus, likely regulates the fusion of endosomes via its interaction with SNAREs (Balderhaar et al., 2013). At present, it is not yet clear how the interaction with SNAREs, lipids, and Vps21 contributes to the fusion process.
Regulation of Ypt7 at the late endosome and vacuole After early endosomes have matured into MVBs/late endosomes, this organelle fuses with the vacuole to deliver its cargo. This requires the recruitment of the fusion machinery to the surface of MVBs, including the Rab7 homolog Ypt7. Recruitment of Ypt7 depends on its GEF, the conserved Mon1-Ccz1 complex (Nordmann et al., 2010;
Gerondopoulos et al., 2012; Cabrera and Ungermann, 2013; Yousefian et al., 2013). Whereas Ypt7 is primarily localized to the vacuolar surface, its GEF Mon1-Ccz1 is mainly localized to endosomes (Nordmann et al., 2010), suggesting that Ypt7 traffics from endosomes to the vacuolar surface. Our data suggest that Mon1-Ccz1 depends on early endosomal fusion factors, including Vps21 (Nordmann et al., 2010), for its recruitment to endosomes, which would be similar to findings on Mon1 and Ccz1 function in metazoan cells (Kinchen and Ravichandran, 2010; Poteryaev et al., 2010). Two GAPs, Msb3 and Gyp7, then act on Ypt7 to convert it back into the inactive GDP-form (Brett et al., 2008; Lachmann et al., 2012; Nickerson et al., 2012), though their timing and function at the vacuole has not been dissected in detail.
The HOPS complex as an effector of Ypt7 Among the characterized effectors of Ypt7 are the HOPS complex and the retromer complex. Whereas the HOPS complex tethers late endosomes and the vacuole during the fusion, the retromer is required for the recycling of receptors of vacuolar hydrolases such as Vps10 receptor from the late endosome back to the Golgi network. It is thought that Ypt7, similar to Rab7 in metazoan cells, binds to retromer prior to the recruitment of HOPS (Rojas et al., 2008; Seaman et al., 2009; Balderhaar et al., 2010; Liu et al., 2012), which would thus allow for efficient receptor recycling before engaging Ypt7 into membrane fusion. The HOPS complex shares the four class C core subunits (Vps11, Vps16, Vps18, Vps33) with the CORVET complex, and has Vps39 and Vps41 as its Rab-specific subunits (Seals et al., 2000; Wurmser et al., 2000). Consequently, HOPS binds efficiently to Ypt7-GTP (Seals et al., 2000; Ostrowicz et al., 2010; Bröcker et al., 2012). The recently published structure of the HOPS complex identified Vps41 and Vps39 at opposite sites of the HOPS complex (Bröcker et al., 2012). This implies that HOPS bridges two Ypt7-positive membranes via two distinct interaction modules, thereby promotes their fusion (Bröcker et al., 2012). HOPS also binds to SNAREs, either in combination or as single SNAREs (Collins et al., 2005; Kramer and Ungermann, 2011; Lobingier and Merz, 2012). This binding seems to be mediated primarily by the SM-protein Vps33, though additional binding sites along the complex have been identified (Kramer and Ungermann, 2011; Lobingier and Merz, 2012). Recent structural analyses of Vps33 in complex with Vps16 fragments demonstrate the predicted
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330 J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling similarity of Vps33 to other Munc18-like proteins and confirm the alpha-helical secondary structure in Vps16 (Baker et al., 2013; Graham et al., 2013). It is not yet clear how HOPS supports SNAREs during fusion, though it likely involves support during their assembly into fusioncompetent complexes (Zick and Wickner, 2013).
Rab5 and Rab7 functions in signaling at endosomes and lysosomes The general role of Rab GTPases in fusion events is well studied. However, Rab GTPases have often been linked to signaling events regulating growth processes of eukaryotic cells (Hutagalung and Novick, 2011). This paragraph will give some insights on the known functions of the endosomal/vacuolar Rab GTPases Vps21 and Ypt7 in comparison to their metazoan homologs Rab5 and Rab7 during processes independent of fusion and their connection to signaling. The target of rapamycin (TOR) is a key regulator of cellular growth processes. This kinase complex is conserved between yeast and metazoans, and exists in two complexes, TORC1 and TORC2 (Durán and Hall, 2012). These complexes are activated by nutrients (TORC1) or by growth factors (TORC2), and thus, promote cell growth and proliferation. In turn, inactivation of TORC1 due to nutrient deprivation results in the onset of autophagy. Whereas TORC2 is required at the plasma membrane (Berchtold and Walther, 2009), TORC1 is found on the surface of vacuoles (Sturgill et al., 2008). Likewise, metazoan TORC1 is recruited to lysosomes by the amino acid sensing Ragulator complex, which is largely equivalent to the yeast EGO complex (Dubouloz et al., 2005; Sancak et al., 2010; Bonfils et al., 2012). This raises the possibility that the maintenance of the lysosome/vacuole homeostasis and thus Rab5 and Rab7 GTPase function is directly linked to TOR signaling (Durán and Hall, 2012). Indeed, depletion of Rab5 in Drosophila or the expression of dominant active Rab5 and Rab7, which interfere with endosomal maturation, results in reduced TORC1 activity (Li et al., 2010). In line with these data, overexpression of Rab5 wild-type or a constitutively active mutant alters localization of mTORC1, and thereby, inhibits the activation by insulin in mammalian cells (Flinn et al., 2010). In yeast cells, deletion of Vps21 or its GEF Vps9 also causes defect in TORC1 activation and hypersensitivity to rapamycin (Bridges et al., 2012a). These data suggest that Rab5 or Rab7 are needed for regulation of TORC1 activity. Interestingly, mutants in the Vps Class C proteins that are
shared between the CORVET and HOPS complex strongly reduce TORC1 signaling (Zurita-Martinez et al., 2007), and the HOPS subunit Vps39/Vam6 has been implicated as a direct modulator of amino acid sensing via the EGO complex (Binda et al., 2009; Valbuena et al., 2012). In combination, these data imply that modulation of the Rabs or their effectors could directly control signaling via the TOR complex. The necessity of active Rab5 for activation of TORC1 may be linked in part to the availability of phosphoinositide-3-phosphates (PI-3-P) (Figure 1, top part). A conserved downstream effector of Rab5 is the PI-3-kinase complex III. This lipid kinase is equivalent to the yeast protein Vps34 and produces PI-3-P, which is needed for the recruitment of additional effectors to endosomal membranes and therefore is essential for proper endosomal maturation (Christoforidis et al., 1999; Shin et al., 2005). Furthermore, metazoan Rab7 binds Vps34 and may thus promote production of PI-3-P on late endosomes (Stein et al., 2003). Yeast cells lacking the only PI-3-kinase Vps34 are sensitive to rapamycin, in agreement with a direct correlation of PI-3-P levels and TORC1 activity (Bridges et al., 2012a). Additionally, metazoan Rab5 is required for the insulinstimulated production of PI-3-P at the plasma membrane (Lodhi et al., 2008), which is indirectly linked to the activation of TORC1 on the lysosome (Bar-Peled and Sabatini, 2012). Moreover, the lysosomal PI-lipid PI-3,5-P2, which is generated from PI-3-P, has been implicated in the localization of TORC1 to lysosomes (Bridges et al., 2012b). Thus, signaling within the endocytic pathway may not only be initiated via Rab5 and Rab7 GTPases, but also directly affect TORC1-driven cellular proliferation.
Outlook: crosstalk signaling and fusion Trafficking and cellular signaling seem to be tightly linked, even though the molecular details of this crosstalk are still largely unknown. The link between TORC1 sig naling and trafficking has so far been studied mainly in deletions, which could perturb both the transport toward endosomes and vacuoles and the vacuole biogenesis. It thus remains unclear if the observed phenotypes are due to the loss of a central lipid, such as PI-3-P or PI-3,5-P2, and a binding platform on endosomes and lysosomes for signaling proteins, or whether endocytic trafficking, per se, is required for TORC1 function. A link between trafficking and signaling could be facilitated by direct regulation or by sharing subunits. Surprisingly, Sec13 has been
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J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling 331
linked to both nuclear pore function, activation of TORC1 and the generation of ER-derived vesicles (Panchaud et al., 2013). Inhibiting Sec13 could thus affect three processes in parallel, which would be an intriguing mode of regulation. However, at this point no evidence for such a regulation has been provided. The close link between the induction of autophagy and TORC1 activity is known in some detail (Chen and Klionsky, 2011), although the parallel effect on general trafficking is only slowly becoming apparent. Thus, we expect that a closer analysis of trafficking processes in light of cellular signaling will
reveal additional insights into the role of Rab5 and Rab7 GTPases in this context. Acknowledgments: We thank Margarita Cabrera for comments and feedback. Research in the Ungermann laboratory is supported by the DFG (UN111/7-2) and the SFB 944 (Project P11). C.U. is supported by the Hans-Mühlenhoff Foundation. Received September 18, 2013; accepted October 22, 2013; previously published online October 23, 2013
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J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling 333 Seaman, M.N., Harbour, M.E., Tattersall, D., Read, E., and Bright, N. (2009). Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5. J. Cell Sci. 122, 2371–2382. Shin, H.W., Hayashi, M., Christoforidis, S., Lacas-Gervais, S., Hoepfner, S., Wenk, M.R., Modregger, J., Uttenweiler-Joseph, S., Wilm, M., Nystuen, A., et al. (2005). An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway. J. Cell Biol. 170, 607–618. Singer-Kruger, B., Stenmark, H., Dusterhoft, A., Philippsen, P., Yoo, J., Gallwitz, D., and Zerial, M. (1994). Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. J. Cell Biol. 125, 283–298. Stein, M.P., Feng, Y., Cooper, K.L., Welford, A.M., and WandingerNess, A. (2003). Human VPS34 and p150 are Rab7 interacting partners. Traffic 4, 754–771. Sturgill, T.W., Cohen, A., Diefenbacher, M., Trautwein, M., Martin, D.E., and Hall, M.N. (2008). TOR1 and TOR2 have distinct locations in live cells. Eukaryotic Cell 7, 1819–1830. Tall, G., Hama, H., DeWald, D., and Horazdovsky, B. (1999). The phosphatidylinositol 3-phosphate binding protein Vac1p
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Minireview Johannes Numrich and Christian Ungermann*
Endocytic Rabs in membrane trafficking and signaling Abstract: The endolysosomal system controls the trafficking of proteins between the plasma membrane and the degradative environment of the lysosome. The early endosomal Rab5 and the late endosomal Rab7 GTPases have a key role in the transport along the endocytic pathway by recruiting tethering factors such as the hexameric CORVET and HOPS complexes that promote membrane fusion. Both Rabs are also involved in signaling at endosomal membranes and linked to amino acid sensing and autophagy, indicating that their role in trafficking may be connected to signal transduction and adaptation during cell stress. Here, we will summarize the current knowledge on the role of both Rab GTPases on both processes and discuss the possible crosstalk between them. Keywords: endosome; Rab5; Rab7; signaling; trafficking; vacuole; Vps21; Ypt7. *Corresponding author: Christian Ungermann, Department of Biology/Chemistry, Biochemistry Section, University of Osnabrück, Barbarastrasse 13, D-49076 Osnabrück, Germany, e-mail: [email protected] Johannes Numrich: Department of Biology/Chemistry, Biochemistry Section, University of Osnabrück, D-49076 Osnabrück, Germany
Introduction Eukaryotic cells have a highly dynamic endomembrane system, in which organelles are interconnected by vesicular carriers that transport both lipids and proteins (Bonifacino and Glick, 2004). Vesicles form by fission at one organelle and may use the cytoskeleton for long-distance trafficking, delivering their content by subsequent fusion with the target membrane. Whereas fission depends on coat proteins, fusion requires the interplay of a conserved machinery that consists of Rab GTPases, tethering factors and SNAREs. Rab GTPases are considered as organelle-specific markers, which recruit tethering factors to the membrane surface. Tethers also bind to incoming vesicles, via either a vesicular Rab GTPase, SNAREs or lipid, and thus, mediate the first contact of membranes. Once the two membranes are close enough, SNAREs on the vesicle and target membranes zipper
together, and provide the energy for fusion of the proximal lipid bilayers. The identification of the basic mechanism of these processes were acknowledged by granting this years the Nobel prize in Medicine or Physiology to Randy Scheckman, James Rothman and Thomas Südhof.
Rab function Rab GTPases are the key regulators of fusion events that occur in the endomembrane system (Barr, 2013; Barr and Lambright, 2010; Hutagalung and Novick, 2011; Itzen and Goody, 2011). The recruitment of a specific Rab determines the identity of each organelle. The activity of a Rab is provided by its cycle between the inactive GDP- and the active GTP-loaded form, which is then able to interact with effectors such as tethers. Rabs are poor GTPases, and should rather be considered as molecular switches that require co-factors for cycling between their different states (Bos et al., 2007; Itzen and Goody, 2011). Within the cytosol, the GDP-bound Rab is kept soluble by a GDP dissociation inhibitor (GDI), which interacts with the C-terminal prenyl anchor and shields the nucleotide-binding pocket of the N-terminal GTPase domain. To be recruited onto membranes, the GDI has to be released. This process may require a GDI displacement factor (GDF), although its general necessity is unclear. Instead, the activator of the Rab, the guanine nucleotide exchange factor (GEF), may be sufficient to displace both GDI and promote nucleotide exchange (Itzen and Goody, 2011). In the GTP-loaded state, Rabs are stably associated to a membrane via their C-terminal prenyl anchor, and can interact with their respective effectors. As a result of their low hydrolysis rate, Rabs depend on GTPase activating proteins (GAPs), which complement the active site and promotes GTP hydrolysis. The Rab-GDP may then be either extracted from the membrane by GDI or undergo activation again (see above references). Within this review, we will focus exclusively on the yeast Rab GTPases Vps21 and Ypt7 and their metazoan homologs Rab5 and Rab7, which are key regulators of endosomal biogenesis, maturation and fusion (Figure 1, bottom part). We will mostly concentrate on observations made in yeast, and will refer to higher eukaryotes in selected examples.
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328 J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling
Figure 1 Endosomal maturation is linked to TORC1 activation. Trafficking from early endosomes to late endosomes and lysosomes is dependent on Rab5 and Rab7 function. These Rabs recruit the tethering complexes CORVET and HOPS to initiate vesicle fusion and the Vps34 Kinase, which produces phosphoinositide-3-phosphates (PI-3-P) at endosomes. PI-3-P in turn is needed for effector recruitment. Loss of Rab5 or Rab7 function leads to altered PI-3-P levels at endosomes, resulting, on the one hand, in defective endosomal maturation, and on the other hand, in reduced TORC1 activity. If PI-3-P levels directly modulate TORC1 activity via recruitment of regulators of TORC1, or indirectly via influencing endosomal maturation and thereby flux of amino acids, remains to be shown. PM, plasma membrane; EV, endocytic vesicle; LE, late endosome; L/V, lysosome/vacuole.
Rab interactions and regulation within the endocytic pathway
Regulation of the Rab5 GTPase Vps21 by GEFs and GAPs
Early endosomes receive cargoes like cell surface receptors or vacuolar hydrolases via vesicular carriers from the trans Golgi network (TGN) or from the plasma membrane. In yeast, the Rab5-like GTPase Vps21 and its effectors, the CORVET tethering complex and the EEA1 homolog Vac1, mediate the fusion of these carriers with the early endosome (Peterson et al., 1999; Tall et al., 1999; Peplowska et al., 2007; Cabrera et al., 2013; Epp and Ungermann, 2013). Our data suggest that Vac1-mediated fusion is primarily required to fuse endocytic vesicles with endosomes (Cabrera et al., 2013). CORVET, as a heterohexamer, has two subunits that bind to Vps21-GTP, and likely acts in homotypic fusion of endosomes (Peplowska et al., 2007; Balderhaar et al., 2013). Early endosomes subsequently mature into Rab7/Ypt7-positive late endosomes, also called MVBs (multivesicular bodies), which finally fuse with vacuoles (Balderhaar and Ungermann, 2013). During this maturation process, ubiquitinated cell surface receptors are sorted into intraluminal vesicles with the help of the ESCRT protein complexes, whereas non-ubiquitinated receptors are recycled back to the plasma membrane. The latter process depends on the Retromer complex (Seaman et al., 2013). Once the surface of a MVB is cleared from all ubiquitinated receptors, the recruitment and activation of the Ypt7 protein and its effector, the hexameric HOPS complex seems to occur downstream and promote tethering and fusion of mature MVBs with vacuoles (see Figure 1).
The Rab5 homolog Vps21 regulates homotypic fusion of endocytic vesicles and heterotypic fusion events of endocytic or TGN-derived vesicles with the early endosome. Beside Vps21, two additional Rab5 homologs named Ypt52 and Ypt53 act at endosomal membranes (Singer-Kruger et al., 1994). Both have redundant roles with Vps21 (Nickerson et al., 2012; Cabrera et al., 2013), and Ypt53 is mainly expressed during environmental stress (Liu et al., 2011; Nickerson et al., 2012). The most important Rab5 homolog Vps21 is activated by its GEF Vps9, which triggers the exchange of GDP to GTP (Hama et al., 1999). Recent studies identified Muk1 as a second protein with GEF activity towards Vps21 (Cabrera et al., 2013; Paulsel et al., 2013). Muk1 is not necessary for proper sorting of the endosomal cargo protein carboxypeptidase S (Cps1) into intraluminal vesicles of MVBs, indicating that it might not be critical for the activation of Vps21 in vivo. In contrast, a vps9 deletion mutant has the same defect in the missorting of Cps1 as a vps21 mutant (Cabrera et al., 2013). However, Muk1 seems to replace Vps9 partially (Cabrera et al., 2013; Paulsel et al., 2013), and Vps21 is only completely mislocalized in the absence of both GEFs (Cabrera and Ungermann, 2013). Thus, two Rab5-GEFs with some overlapping function are required in yeast, similar to the situation in metazoan cells, where multiple Rab5-GEFs have been identified (Carney et al., 2006). Vps21 is subsequently inactivated by the GAP protein Msb3 (Lachmann et al., 2012; Nickerson et al.,
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J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling 329
2012). Interestingly, Msb3 has been identified as the GAP of the secretory Rab Sec4 (Gao et al., 2003), and requires the heterohexameric BLOC-1 complex for its targeting to endosomal membranes (John Peter et al., 2013). Furthermore, Msb3 has also activity for the Rab7-like Ypt7 protein of the vacuole (Lachmann et al., 2012; Nickerson et al., 2012), suggesting that Msb3 controls the rate of endocytic transport by inactivation of multiple Rabs.
Vps21 effectors Once activated, Vps21 recruits its effectors, the EEA1-like Vac1 and the hexameric CORVET complex, to the endosomal surface. Vac1 belongs to the family of so-called coiled coil tethers, seems to function upstream of CORVET (Cabrera et al., 2013), and is needed for the fusion of endocytic vesicles (Peterson et al., 1999; Tall et al., 1999), whereas the CORVET complex is likely required for homotypic fusion of early endosomes (Balderhaar et al., 2013). The hexameric CORVET complex consists of the four class C subunits (Vps11, Vps16, Vps18 and the Sec1/Munc18 homolog Vps33) and two additional subunits named Vps3 and Vps8 (Peplowska et al., 2007). All subunits except Vps33 have structural homology, and contain a predicted N-terminal b-propeller and a C-terminal α-solenoid domain (Nickerson et al., 2009). The subunits Vps3 and Vps8 interact with the Rab GTPase Vps21 directly (Horazdovsky et al., 1996; Markgraf et al., 2009; Pawelec et al., 2010; Plemel et al., 2011; Epp and Ungermann, 2013). Whether both or only one subunit is needed for the recruitment of the CORVET to endosomes remains unclear (Epp and Ungermann, 2013). Once recruited to the membrane, CORVET promotes tethering of endosomal membranes, and thus, likely regulates the fusion of endosomes via its interaction with SNAREs (Balderhaar et al., 2013). At present, it is not yet clear how the interaction with SNAREs, lipids, and Vps21 contributes to the fusion process.
Regulation of Ypt7 at the late endosome and vacuole After early endosomes have matured into MVBs/late endosomes, this organelle fuses with the vacuole to deliver its cargo. This requires the recruitment of the fusion machinery to the surface of MVBs, including the Rab7 homolog Ypt7. Recruitment of Ypt7 depends on its GEF, the conserved Mon1-Ccz1 complex (Nordmann et al., 2010;
Gerondopoulos et al., 2012; Cabrera and Ungermann, 2013; Yousefian et al., 2013). Whereas Ypt7 is primarily localized to the vacuolar surface, its GEF Mon1-Ccz1 is mainly localized to endosomes (Nordmann et al., 2010), suggesting that Ypt7 traffics from endosomes to the vacuolar surface. Our data suggest that Mon1-Ccz1 depends on early endosomal fusion factors, including Vps21 (Nordmann et al., 2010), for its recruitment to endosomes, which would be similar to findings on Mon1 and Ccz1 function in metazoan cells (Kinchen and Ravichandran, 2010; Poteryaev et al., 2010). Two GAPs, Msb3 and Gyp7, then act on Ypt7 to convert it back into the inactive GDP-form (Brett et al., 2008; Lachmann et al., 2012; Nickerson et al., 2012), though their timing and function at the vacuole has not been dissected in detail.
The HOPS complex as an effector of Ypt7 Among the characterized effectors of Ypt7 are the HOPS complex and the retromer complex. Whereas the HOPS complex tethers late endosomes and the vacuole during the fusion, the retromer is required for the recycling of receptors of vacuolar hydrolases such as Vps10 receptor from the late endosome back to the Golgi network. It is thought that Ypt7, similar to Rab7 in metazoan cells, binds to retromer prior to the recruitment of HOPS (Rojas et al., 2008; Seaman et al., 2009; Balderhaar et al., 2010; Liu et al., 2012), which would thus allow for efficient receptor recycling before engaging Ypt7 into membrane fusion. The HOPS complex shares the four class C core subunits (Vps11, Vps16, Vps18, Vps33) with the CORVET complex, and has Vps39 and Vps41 as its Rab-specific subunits (Seals et al., 2000; Wurmser et al., 2000). Consequently, HOPS binds efficiently to Ypt7-GTP (Seals et al., 2000; Ostrowicz et al., 2010; Bröcker et al., 2012). The recently published structure of the HOPS complex identified Vps41 and Vps39 at opposite sites of the HOPS complex (Bröcker et al., 2012). This implies that HOPS bridges two Ypt7-positive membranes via two distinct interaction modules, thereby promotes their fusion (Bröcker et al., 2012). HOPS also binds to SNAREs, either in combination or as single SNAREs (Collins et al., 2005; Kramer and Ungermann, 2011; Lobingier and Merz, 2012). This binding seems to be mediated primarily by the SM-protein Vps33, though additional binding sites along the complex have been identified (Kramer and Ungermann, 2011; Lobingier and Merz, 2012). Recent structural analyses of Vps33 in complex with Vps16 fragments demonstrate the predicted
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330 J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling similarity of Vps33 to other Munc18-like proteins and confirm the alpha-helical secondary structure in Vps16 (Baker et al., 2013; Graham et al., 2013). It is not yet clear how HOPS supports SNAREs during fusion, though it likely involves support during their assembly into fusioncompetent complexes (Zick and Wickner, 2013).
Rab5 and Rab7 functions in signaling at endosomes and lysosomes The general role of Rab GTPases in fusion events is well studied. However, Rab GTPases have often been linked to signaling events regulating growth processes of eukaryotic cells (Hutagalung and Novick, 2011). This paragraph will give some insights on the known functions of the endosomal/vacuolar Rab GTPases Vps21 and Ypt7 in comparison to their metazoan homologs Rab5 and Rab7 during processes independent of fusion and their connection to signaling. The target of rapamycin (TOR) is a key regulator of cellular growth processes. This kinase complex is conserved between yeast and metazoans, and exists in two complexes, TORC1 and TORC2 (Durán and Hall, 2012). These complexes are activated by nutrients (TORC1) or by growth factors (TORC2), and thus, promote cell growth and proliferation. In turn, inactivation of TORC1 due to nutrient deprivation results in the onset of autophagy. Whereas TORC2 is required at the plasma membrane (Berchtold and Walther, 2009), TORC1 is found on the surface of vacuoles (Sturgill et al., 2008). Likewise, metazoan TORC1 is recruited to lysosomes by the amino acid sensing Ragulator complex, which is largely equivalent to the yeast EGO complex (Dubouloz et al., 2005; Sancak et al., 2010; Bonfils et al., 2012). This raises the possibility that the maintenance of the lysosome/vacuole homeostasis and thus Rab5 and Rab7 GTPase function is directly linked to TOR signaling (Durán and Hall, 2012). Indeed, depletion of Rab5 in Drosophila or the expression of dominant active Rab5 and Rab7, which interfere with endosomal maturation, results in reduced TORC1 activity (Li et al., 2010). In line with these data, overexpression of Rab5 wild-type or a constitutively active mutant alters localization of mTORC1, and thereby, inhibits the activation by insulin in mammalian cells (Flinn et al., 2010). In yeast cells, deletion of Vps21 or its GEF Vps9 also causes defect in TORC1 activation and hypersensitivity to rapamycin (Bridges et al., 2012a). These data suggest that Rab5 or Rab7 are needed for regulation of TORC1 activity. Interestingly, mutants in the Vps Class C proteins that are
shared between the CORVET and HOPS complex strongly reduce TORC1 signaling (Zurita-Martinez et al., 2007), and the HOPS subunit Vps39/Vam6 has been implicated as a direct modulator of amino acid sensing via the EGO complex (Binda et al., 2009; Valbuena et al., 2012). In combination, these data imply that modulation of the Rabs or their effectors could directly control signaling via the TOR complex. The necessity of active Rab5 for activation of TORC1 may be linked in part to the availability of phosphoinositide-3-phosphates (PI-3-P) (Figure 1, top part). A conserved downstream effector of Rab5 is the PI-3-kinase complex III. This lipid kinase is equivalent to the yeast protein Vps34 and produces PI-3-P, which is needed for the recruitment of additional effectors to endosomal membranes and therefore is essential for proper endosomal maturation (Christoforidis et al., 1999; Shin et al., 2005). Furthermore, metazoan Rab7 binds Vps34 and may thus promote production of PI-3-P on late endosomes (Stein et al., 2003). Yeast cells lacking the only PI-3-kinase Vps34 are sensitive to rapamycin, in agreement with a direct correlation of PI-3-P levels and TORC1 activity (Bridges et al., 2012a). Additionally, metazoan Rab5 is required for the insulinstimulated production of PI-3-P at the plasma membrane (Lodhi et al., 2008), which is indirectly linked to the activation of TORC1 on the lysosome (Bar-Peled and Sabatini, 2012). Moreover, the lysosomal PI-lipid PI-3,5-P2, which is generated from PI-3-P, has been implicated in the localization of TORC1 to lysosomes (Bridges et al., 2012b). Thus, signaling within the endocytic pathway may not only be initiated via Rab5 and Rab7 GTPases, but also directly affect TORC1-driven cellular proliferation.
Outlook: crosstalk signaling and fusion Trafficking and cellular signaling seem to be tightly linked, even though the molecular details of this crosstalk are still largely unknown. The link between TORC1 sig naling and trafficking has so far been studied mainly in deletions, which could perturb both the transport toward endosomes and vacuoles and the vacuole biogenesis. It thus remains unclear if the observed phenotypes are due to the loss of a central lipid, such as PI-3-P or PI-3,5-P2, and a binding platform on endosomes and lysosomes for signaling proteins, or whether endocytic trafficking, per se, is required for TORC1 function. A link between trafficking and signaling could be facilitated by direct regulation or by sharing subunits. Surprisingly, Sec13 has been
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J. Numrich and C. Ungermann: Endocytic Rabs in membrane trafficking and signaling 331
linked to both nuclear pore function, activation of TORC1 and the generation of ER-derived vesicles (Panchaud et al., 2013). Inhibiting Sec13 could thus affect three processes in parallel, which would be an intriguing mode of regulation. However, at this point no evidence for such a regulation has been provided. The close link between the induction of autophagy and TORC1 activity is known in some detail (Chen and Klionsky, 2011), although the parallel effect on general trafficking is only slowly becoming apparent. Thus, we expect that a closer analysis of trafficking processes in light of cellular signaling will
reveal additional insights into the role of Rab5 and Rab7 GTPases in this context. Acknowledgments: We thank Margarita Cabrera for comments and feedback. Research in the Ungermann laboratory is supported by the DFG (UN111/7-2) and the SFB 944 (Project P11). C.U. is supported by the Hans-Mühlenhoff Foundation. Received September 18, 2013; accepted October 22, 2013; previously published online October 23, 2013
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