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Ypt1 and COPII vesicles act in autophagosome biogenesis and the early secretory pathway Saralin Davis* and Susan Ferro-Novick*1 *Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093-0668, U.S.A.
Abstract The GTPase Ypt1, Rab1 in mammals functions on multiple intracellular trafficking pathways. Ypt1 has an established role on the early secretory pathway in targeting coat protein complex II (COPII) coated vesicles to the cis-Golgi. Additionally, Ypt1 functions during the initial stages of macroautophagy, a process of cellular degradation induced during periods of cell stress. In the present study, we discuss the role of Ypt1 and other secretory machinery during macroautophagy, highlighting commonalities between these two pathways.
Introduction Macroautophagy is a catabolic process designed to rid the cell of unwanted proteins and organelles. During cell stress macroautophagy is up-regulated leading to a dramatic reorganization of membranes needed to form the double membrane structure called the autophagosome, which targets proteins and organelles for bulk degradation [1]. When macrautophagy is initiated, secretion slows down [2], indicating a proper balance of these events is needed. Recent findings have revealed that a part of the machinery required for autophagosome formation is shared with that of the secretory pathway. Our understanding of how classical trafficking machinery participates in macroautophagy has begun to shed light on the mechanism of autophagosome biogenesis. Membrane trafficking requires precise co-ordination of vesicle budding, tethering and fusion to ensure cargoes and lipids are targeted to the correct intracellular destination. Rab GTPases, members of the Ras superfamily of GTPases, play a central role in regulating membrane-trafficking events [3]. There are 11 Rabs in yeast and over 60 Rabs in mammalian cells that function at different membranes in order to regulate the various transport stages [4]. GTPases act as molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound form. GTPases have an inherently low intrinsic rate of GDP-dissociation and consequently guaninenucleotide-exchange factors (GEFs) are needed to catalyse the switch from the GDP to GTP-bound form. Once in an active GTP state, the Rab is inserted into the membranes through a prenyl group on C-terminal cysteines. In the present study, the activated Rab recruits specific effector proteins that carry out various trafficking duties. Rabs are inactivated by GTPactivating proteins (GAPs), which catalyse GTP hydrolysis. Key words: COPII vesicle, macroautophagy, membrane trafficking, TRAPP complex, Ypt1. Abbreviations: Atg, autophagy-related; COPII, coat protein complex II; Cvt, cytoplasmic to vacuolar targeting; ER, endoplasmic reticulum; GAP, GTP-activating protein; GEF, guaninenucleotide-exchange factor; PAS, pre-autophagosomal site; SNARE, soluble N-ethylmaleimidesensitive factor-attachment protein receptor; TRAPP, transport particle protein; t-SNARE, target SNARE. 1 To whom correspondence should be addressed (email [email protected]).
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This GTP cycle allows for the temporal and spatial control of effector protein recruitment [4]. The GTPase Ypt1 (Rab1 in mammals) functions in multiple trafficking events. Ypt1 is essential for transport from the endoplasmic reticulum (ER) to the cis-Golgi, intra-Golgi transport and macroautophagy. Three distinct forms of the multimeric GEF transport particle protein (TRAPP) activate Ypt1 on each of these pathways: TRAPPI (ER–Golgi), TRAPPII (intra-Golgi) and TRAPPIII (macroautophagy) [5]. TRAPPI contains six subunits (Bet3, Bet5, Trs20, Trs23, Trs31, Trs33) that are shared with TRAPPII and TRAPPIII. Four of these subunits (Bet3, Bet5, Trs23 and Trs31) are essential for GEF activity [6]. TRAPPII and TRAPPIII contain additional unique subunits that redirect the complex to their respective pathways. TRAPPII contains the unique subunits Trs65, Trs120 and Trs130 that redirect TRAPPII to the Golgi [7–9]. TRAPPIII contains the unique Trs85 subunit, which was recently reported to interact with machinery on the macroautophagy pathway [10,11]. In mammalian cells two TRAPP complexes have been identified, equivalent to yeast TRAPPII and TRAPPIII [8,10,12]. Mammalian TRAPP complexes also activate Rab1 and the functions of the complexes appear to be highly conserved [8,10]. In the present paper, we describe recent findings that have clarified the role of TRAPPIII and Ypt1 during macroautophagy, which has helped contribute to our understanding of autophagosome formation.
Ypt1 and TRAPPI in ER to Golgi transport The function of Ypt1 has been most studied on the ER to Golgi pathway where Ypt1 helps target coat protein complex II (COPII) coated vesicles to the cis-Golgi. Another GTPase, Sar1, initiates COPII vesicle formation at specialized domains of the ER called ER exit sites. Sar1–GTP becomes anchored to the ER membrane where it induces membrane curvature and recruits the inner COPII coat, Sec23/Sec24. Sec23/Sec24 then recruits the outer coat shell, Sec13/Sec31, which leads to coat polymerization and vesicle budding Biochem. Soc. Trans. (2015) 43, 92–96; doi:10.1042/BST20140247
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Figure 1 Model of COPII vesicle transport from the ER to the Golgi COPII coat formation is initiated at ER-exit sites by activated Sar1. Sar1 recruits the Sec23/Sec24 complex, which then leads to the recruitment of the Sec13/Sec31 complex and vesicle budding. Sar1 is hydrolysed and released from the vesicle, which exposes a binding site on Sec23 for TRAPPI (1). TRAPPI recruits and activates Ypt1/Rab1 (2). Activated Ypt1/Rab1 recruits Uso1/p115 (mammalian Uso1), which links the vesicle to the cis-Golgi (3). At the Golgi, Hrr25 or CKIδ in mammalian cells displaces TRAPPI from the vesicle and phosphorylates Sec23 (4). SNARE pairing and membrane fusion proceed after the vesicle uncoats (5). Sit4 or PP6 in mammalian cells dephosphorylates the coat, allowing for a new round of vesicle budding (6).
[13]. Sec23 is the GAP for Sar1 and the GAP activity of Sec23 is enhanced by Sec31 [14]. As a result, once the full coat has formed Sar1 is inactivated and released from the vesicle. Release of Sar1 exposes a binding site on Sec23 for the TRAPPI subunit Bet3 (Figure 1, stage 1). TRAPPI binds to Sec23 and recruits and activates Ypt1 on the vesicle [15] (Figure 1, stage 2). Ypt1–GTP then recruits its effector the long coiled-coil tether Uso1, which physically links the vesicle to the cis-Golgi [16] (Figure 1, stage 3). At the Golgi, a serine/threonine kinase, Hrr25 displaces TRAPPI from the vesicle and phosphorylates Sec23 [17] (Figure 1, stage 4). Thus, through a series of sequential interactions with Sar1–GTP, TRAPPI and Hrr25, Sec23 provides directionality to COPII vesicle transport [17]. At the Golgi, the vesicle must uncoat to allow for soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) pairing of a vesicle SNARE (v-SNARE) on a vesicle with a target SNARE (t-SNARE) complex at the target membrane. Formation of this SNARE complex drives vesicle fusion (Figure 1, stage 5). Upon uncoating, the phosphorylated coat is released into the cytosol and dephosphroylated by the phosphatase Sit4, which recycles the coat for a new round of vesicle budding at the ER [18] (Figure 1, stage 6). In mammalian cells, COPII vesicles do not directly tether with the Golgi, rather they undergo homotypic tethering to form vesicular tubular clusters (VTCs), which is a preGolgi compartment that may be equivalent to the early Golgi
in yeast. Despite this difference, the events described above appear to be conserved. Sec23 and its three different binding partners are all required for ER–Golgi traffic in mammals and appear to function in a similar manner [17,18]. The mammalian homologue of Bet3, TRAPPC3, is required for homotypic tethering of COPII vesicles and directly interacts with mammalian Sec23 (mSec23) [15,19]. Additionally, activated Rab1 recruits the mammalian homologue of Uso1, p115 to vesicles [20]. p115 has also been reported to bind to the t-SNARE syntaxin-5 on the cis-Golgi. Binding of p115 to syntaxin-5 is critical for p115 localization and is thought to help catalyse SNARE complex formation [21,22]. Therefore, through its effector p115, Rab1 is able to co-ordinate COPII vesicle tethering and fusion.
Ypt1 and TRAPPIII in macroautophagy Recent findings have begun to reveal the role of Ypt1 in macroautophagy, a process of bulk degradation used by the cell during nutrient-limiting conditions [10,11,29]. When macroautophagy is induced, a double membrane called the phagophore forms at a pre-autophagosomal site (PAS). The phagophore then extends and seals to form the double membrane autophagosome. The autophagosome subsequently fuses with the vacuole, releasing its contents for degradation [1]. The PAS is situated between the vacuole and ER-exit sites and autophagy-related (Atg) proteins localize C The
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to the PAS in a hierarchical manner [23–25]. The most upstream protein complex in PAS assembly is the Atg1 initiating complex. The scaffold protein Atg17 forms a stable complex with Atg29 and Atg31 that interacts with Atg1 and Atg13 when macroautophagy is induced [26,27]. Together Atg17 and Atg13 activate Atg1 kinase activity [28]. This upstream complex is needed for recruitment of downstream Atg proteins and autophagosome formation. Ypt1 and its autophagy-specific GEF TRAPPIII also act at this early stage of phagophore initiation. Atg17 recruits TRAPPIII to the PAS by interacting with the TRAPPIII subunit Trs85 [10]. TRAPPIII then recruits and activates Ypt1 at the PAS [11]. A screen in yeast revealed Atg1 is an Ypt1 effector [10]. Although Ypt1 does not regulate Atg1 kinase activity, it is required to efficiently recruit Atg1 to the PAS [10]. As Atg1 is a key driver of macroautophagy, having multiple regulators of Atg1 recruitment is probably needed for proper mediation of the autophagy response. The single-particle EM structure of the autophagy-specific GEF, TRAPPIII, showed the Trs85 subunit is located at the end of the elongated TRAPPIII structure [29]. The positioning of Trs85 leaves Bet3 accessible for Sec23 binding suggesting that, like TRAPPI, TRAPPIII recruits Ypt1 to COPII vesicles [29]. This finding is consistent with a growing body of work implicating COPII vesicles in autophagosome formation.
COPII vesicles in autophagosome formation Although our understanding of the assembly of Atg proteins at the PAS has advanced significantly over the last two decades, the membrane trafficking events leading to autophagosome formation remain unclear. In yeast, two classes of vesicles, Atg9 and COPII, have been implicated in this process. Atg9 vesicles are trafficked from the transGolgi to the PAS and are required just downstream of the Atg1-initiating complex [30]. Although Atg9 vesicles are essential for phagophore initiation, only a few Atg9 vesicles are needed, indicating other membranes are involved [30]. COPII coat subunits were reported early on to be required for autophagosome formation [31,32]. These studies postulated that general flux of traffic from the ER might be needed for macroautophagy rather than a direct involvement of COPII vesicles [31]. Recent work, however, has begun to favour a model in which COPII vesicles directly contribute to autophagosome biogenesis [21,22,29]. The observation that the PAS is formed adjacent to ER-exit sites is suggestive of a role of COPII vesicles in autophagosome formation [24,25]. Consistent with this notion, when macroautophagy is blocked, COPII vesicles begin to accumulate at the PAS [29]. A recent proteomic study identified physical interactions between COPII coat subunits and many Atg proteins. The study by Graef et al. [24] went on to demonstrate COPII vesicles are required just downstream of the Atg1 initiating complex; however, the exact function of COPII vesicles at this step has yet to be determined. Moreover, many regulators of ER to Golgi trafficking, including Sar1 and the ER–Golgi SNAREs, were shown to be required for macroautophagy C The
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[29]. Notably, the only exception was the tether Uso1 [29]. This observation supports the notion that COPII vesicles are not indirectly playing a role in macroautophagy by transporting cargo to the Golgi. Furthermore, it raises the model that COPII vesicles are transported by a similar mechanism during macroautophagy, except a different tether targets COPII vesicles to a different acceptor compartment. The serine/threonine kinase Atg1 is the likely tether. Atg1 can directly bind membranes in vitro, which is consistent with reports that Atg1 has kinase-independent functions required upstream of Atg1 kinase activity [33,34]. Atg1 also directly binds to and phosphorylates Atg9 making it tempting to speculate Atg1, may tether Atg9 and COPII vesicles, although this model awaits further validation [35] (Figure 2).
Rab1 and mTRAPPIII in macroautophagy Although our understanding of the hierarchy of Atg proteins in the mammalian cells is less clear, these events appear to be generally conserved. In the mammalian cells, there is not just a single PAS, but multiple sites at which autophagosome formation is initiated. These sites have been localized to the ER and more specifically to the ER-exit sites [24,29]. Autophagosome formation in mammalian cells probably also involves Atg9 and COPII vesicles [36], although additional membrane sources, including ER–mitochondria contact sites, the mitochondria, the ER–Golgi intermediate compartment (ERGIC) and the plasma membrane, have also been implicated [37–40]. Rab1b and mammalian TRAPPIII are also required for autophagosome biogenesis [41,42]. Furthermore, Rab1 was reported to interact in vivo with Ulk1, the mammalian homologue of Atg1 [10]. Ulk1 forms a complex with the Atg17-like protein FIP200 [43,44]. Further work will be needed to verify whether FIP200 plays a similar role in recruiting mammalian TRAPPIII and Rab1 to sites of autophagosome formation.
Ypt1 and TRAPPIII during selective autophagy In addition to bulk macroautophagy, there exist several forms of selective autophagy that transport specific cargo, such as damaged organelles, for vacuolar degradation. Selective autophagy shares some of the same machinery as macroautophagy; however, as the pathways become better understood, clear differences have emerged. In yeast, the most well-studied form of selective autophagy is the cytoplasmic to vacuolar targeting (Cvt) pathway through which various vacuolar hydrolases are transported to the vacuole for activation. Ypt1 and TRAPPIII are both required for the Cvt pathway and may perform distinct functions from their roles during macroautophagy [11,45]. In nutrient-rich conditions, Ypt1 was reported to have a different effector, the Cvt-specific scaffold Atg11 [46]. Atg11 acts upstream in recruiting Atg proteins to the PAS for selective autophagy. Although Atg9 vesicles are required for both selective and macroautophagy
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Figure 2 Model of early autophagosome formation Upon induction of macroautophagy, the PAS is formed adjacent to the ER-exit sites and the vacuole. At the PAS, the Atg17 complex recruits TRAPPIII. TRAPPIII binds to the Sec23 subunit of the COPII coat and recruits and activates Ypt1. Activated Ypt1 aids in recruitment of Atg1. Atg1 binds and phosphorylates Atg9 and may tether Atg9 and COPII vesicles, initiating autophagosome formation.
[47], COPII vesicles do not seem to be required for the Cvt pathway [31]. TRAPPIII has been implicated in the transport of Atg9 vesicles from the endosome to the Golgi and this function is more essential in nutrient-rich conditions [48]. Ypt1 and TRAPPIII are probably involved in other forms of selective autophagy as Trs85 is required for the selective autophagy of peroxisomes and Rab1 and mammalian TRAPPIII were found to be required for antimicrobial autophagy [45,49]. Additional work will be needed to determine the role of Ypt1 during these other types of selective autophagy.
Concluding remarks The formation of the autophagosome has received significant attention in recent years resulting in a surge of new findings on this previously mysterious process. Work on the structure and function of Ypt1 and TRAPPIII have highlighted commonalities between the ER to Golgi pathway and the early stages of autophagosome biogenesis, namely the involvement of COPII vesicles. Although we have a good grasp of how directionality is achieved on the ER to Golgi pathway, our understanding of how COPII vesicles are redirected to the PAS to aid in autophagosome formation is unknown. As the tether Uso1 is not required for macroautophagy, tethering is probably an important decision point for the fate of COPII vesicles. It is also possible that the ER to Golgi trafficking machinery is modified during macroautophagy to preferentially interact with Atg proteins or to inhibit interactions with secretory machinery. Finally, COPII vesicles may be passively redirected to the PAS as the PAS is formed adjacent to ER-exit sites. This may allow the macroautophagy pathway to outcompete the secretory pathway for access to COPII vesicles. Understanding how the PAS is formed at ER-exit sites as well as obtaining a
better grasp of vesicle tethering and fusion events during phagophore initiation will be required in order to address these key questions.
Acknowledgements Salary support was provided by the Howard Hughes Medical Institute.
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Received 17 September 2014 doi:10.1042/BST20140247
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Ypt1 and COPII vesicles act in autophagosome biogenesis and the early secretory pathway Saralin Davis* and Susan Ferro-Novick*1 *Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093-0668, U.S.A.
Abstract The GTPase Ypt1, Rab1 in mammals functions on multiple intracellular trafficking pathways. Ypt1 has an established role on the early secretory pathway in targeting coat protein complex II (COPII) coated vesicles to the cis-Golgi. Additionally, Ypt1 functions during the initial stages of macroautophagy, a process of cellular degradation induced during periods of cell stress. In the present study, we discuss the role of Ypt1 and other secretory machinery during macroautophagy, highlighting commonalities between these two pathways.
Introduction Macroautophagy is a catabolic process designed to rid the cell of unwanted proteins and organelles. During cell stress macroautophagy is up-regulated leading to a dramatic reorganization of membranes needed to form the double membrane structure called the autophagosome, which targets proteins and organelles for bulk degradation [1]. When macrautophagy is initiated, secretion slows down [2], indicating a proper balance of these events is needed. Recent findings have revealed that a part of the machinery required for autophagosome formation is shared with that of the secretory pathway. Our understanding of how classical trafficking machinery participates in macroautophagy has begun to shed light on the mechanism of autophagosome biogenesis. Membrane trafficking requires precise co-ordination of vesicle budding, tethering and fusion to ensure cargoes and lipids are targeted to the correct intracellular destination. Rab GTPases, members of the Ras superfamily of GTPases, play a central role in regulating membrane-trafficking events [3]. There are 11 Rabs in yeast and over 60 Rabs in mammalian cells that function at different membranes in order to regulate the various transport stages [4]. GTPases act as molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound form. GTPases have an inherently low intrinsic rate of GDP-dissociation and consequently guaninenucleotide-exchange factors (GEFs) are needed to catalyse the switch from the GDP to GTP-bound form. Once in an active GTP state, the Rab is inserted into the membranes through a prenyl group on C-terminal cysteines. In the present study, the activated Rab recruits specific effector proteins that carry out various trafficking duties. Rabs are inactivated by GTPactivating proteins (GAPs), which catalyse GTP hydrolysis. Key words: COPII vesicle, macroautophagy, membrane trafficking, TRAPP complex, Ypt1. Abbreviations: Atg, autophagy-related; COPII, coat protein complex II; Cvt, cytoplasmic to vacuolar targeting; ER, endoplasmic reticulum; GAP, GTP-activating protein; GEF, guaninenucleotide-exchange factor; PAS, pre-autophagosomal site; SNARE, soluble N-ethylmaleimidesensitive factor-attachment protein receptor; TRAPP, transport particle protein; t-SNARE, target SNARE. 1 To whom correspondence should be addressed (email [email protected]).
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This GTP cycle allows for the temporal and spatial control of effector protein recruitment [4]. The GTPase Ypt1 (Rab1 in mammals) functions in multiple trafficking events. Ypt1 is essential for transport from the endoplasmic reticulum (ER) to the cis-Golgi, intra-Golgi transport and macroautophagy. Three distinct forms of the multimeric GEF transport particle protein (TRAPP) activate Ypt1 on each of these pathways: TRAPPI (ER–Golgi), TRAPPII (intra-Golgi) and TRAPPIII (macroautophagy) [5]. TRAPPI contains six subunits (Bet3, Bet5, Trs20, Trs23, Trs31, Trs33) that are shared with TRAPPII and TRAPPIII. Four of these subunits (Bet3, Bet5, Trs23 and Trs31) are essential for GEF activity [6]. TRAPPII and TRAPPIII contain additional unique subunits that redirect the complex to their respective pathways. TRAPPII contains the unique subunits Trs65, Trs120 and Trs130 that redirect TRAPPII to the Golgi [7–9]. TRAPPIII contains the unique Trs85 subunit, which was recently reported to interact with machinery on the macroautophagy pathway [10,11]. In mammalian cells two TRAPP complexes have been identified, equivalent to yeast TRAPPII and TRAPPIII [8,10,12]. Mammalian TRAPP complexes also activate Rab1 and the functions of the complexes appear to be highly conserved [8,10]. In the present paper, we describe recent findings that have clarified the role of TRAPPIII and Ypt1 during macroautophagy, which has helped contribute to our understanding of autophagosome formation.
Ypt1 and TRAPPI in ER to Golgi transport The function of Ypt1 has been most studied on the ER to Golgi pathway where Ypt1 helps target coat protein complex II (COPII) coated vesicles to the cis-Golgi. Another GTPase, Sar1, initiates COPII vesicle formation at specialized domains of the ER called ER exit sites. Sar1–GTP becomes anchored to the ER membrane where it induces membrane curvature and recruits the inner COPII coat, Sec23/Sec24. Sec23/Sec24 then recruits the outer coat shell, Sec13/Sec31, which leads to coat polymerization and vesicle budding Biochem. Soc. Trans. (2015) 43, 92–96; doi:10.1042/BST20140247
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Figure 1 Model of COPII vesicle transport from the ER to the Golgi COPII coat formation is initiated at ER-exit sites by activated Sar1. Sar1 recruits the Sec23/Sec24 complex, which then leads to the recruitment of the Sec13/Sec31 complex and vesicle budding. Sar1 is hydrolysed and released from the vesicle, which exposes a binding site on Sec23 for TRAPPI (1). TRAPPI recruits and activates Ypt1/Rab1 (2). Activated Ypt1/Rab1 recruits Uso1/p115 (mammalian Uso1), which links the vesicle to the cis-Golgi (3). At the Golgi, Hrr25 or CKIδ in mammalian cells displaces TRAPPI from the vesicle and phosphorylates Sec23 (4). SNARE pairing and membrane fusion proceed after the vesicle uncoats (5). Sit4 or PP6 in mammalian cells dephosphorylates the coat, allowing for a new round of vesicle budding (6).
[13]. Sec23 is the GAP for Sar1 and the GAP activity of Sec23 is enhanced by Sec31 [14]. As a result, once the full coat has formed Sar1 is inactivated and released from the vesicle. Release of Sar1 exposes a binding site on Sec23 for the TRAPPI subunit Bet3 (Figure 1, stage 1). TRAPPI binds to Sec23 and recruits and activates Ypt1 on the vesicle [15] (Figure 1, stage 2). Ypt1–GTP then recruits its effector the long coiled-coil tether Uso1, which physically links the vesicle to the cis-Golgi [16] (Figure 1, stage 3). At the Golgi, a serine/threonine kinase, Hrr25 displaces TRAPPI from the vesicle and phosphorylates Sec23 [17] (Figure 1, stage 4). Thus, through a series of sequential interactions with Sar1–GTP, TRAPPI and Hrr25, Sec23 provides directionality to COPII vesicle transport [17]. At the Golgi, the vesicle must uncoat to allow for soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) pairing of a vesicle SNARE (v-SNARE) on a vesicle with a target SNARE (t-SNARE) complex at the target membrane. Formation of this SNARE complex drives vesicle fusion (Figure 1, stage 5). Upon uncoating, the phosphorylated coat is released into the cytosol and dephosphroylated by the phosphatase Sit4, which recycles the coat for a new round of vesicle budding at the ER [18] (Figure 1, stage 6). In mammalian cells, COPII vesicles do not directly tether with the Golgi, rather they undergo homotypic tethering to form vesicular tubular clusters (VTCs), which is a preGolgi compartment that may be equivalent to the early Golgi
in yeast. Despite this difference, the events described above appear to be conserved. Sec23 and its three different binding partners are all required for ER–Golgi traffic in mammals and appear to function in a similar manner [17,18]. The mammalian homologue of Bet3, TRAPPC3, is required for homotypic tethering of COPII vesicles and directly interacts with mammalian Sec23 (mSec23) [15,19]. Additionally, activated Rab1 recruits the mammalian homologue of Uso1, p115 to vesicles [20]. p115 has also been reported to bind to the t-SNARE syntaxin-5 on the cis-Golgi. Binding of p115 to syntaxin-5 is critical for p115 localization and is thought to help catalyse SNARE complex formation [21,22]. Therefore, through its effector p115, Rab1 is able to co-ordinate COPII vesicle tethering and fusion.
Ypt1 and TRAPPIII in macroautophagy Recent findings have begun to reveal the role of Ypt1 in macroautophagy, a process of bulk degradation used by the cell during nutrient-limiting conditions [10,11,29]. When macroautophagy is induced, a double membrane called the phagophore forms at a pre-autophagosomal site (PAS). The phagophore then extends and seals to form the double membrane autophagosome. The autophagosome subsequently fuses with the vacuole, releasing its contents for degradation [1]. The PAS is situated between the vacuole and ER-exit sites and autophagy-related (Atg) proteins localize C The
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to the PAS in a hierarchical manner [23–25]. The most upstream protein complex in PAS assembly is the Atg1 initiating complex. The scaffold protein Atg17 forms a stable complex with Atg29 and Atg31 that interacts with Atg1 and Atg13 when macroautophagy is induced [26,27]. Together Atg17 and Atg13 activate Atg1 kinase activity [28]. This upstream complex is needed for recruitment of downstream Atg proteins and autophagosome formation. Ypt1 and its autophagy-specific GEF TRAPPIII also act at this early stage of phagophore initiation. Atg17 recruits TRAPPIII to the PAS by interacting with the TRAPPIII subunit Trs85 [10]. TRAPPIII then recruits and activates Ypt1 at the PAS [11]. A screen in yeast revealed Atg1 is an Ypt1 effector [10]. Although Ypt1 does not regulate Atg1 kinase activity, it is required to efficiently recruit Atg1 to the PAS [10]. As Atg1 is a key driver of macroautophagy, having multiple regulators of Atg1 recruitment is probably needed for proper mediation of the autophagy response. The single-particle EM structure of the autophagy-specific GEF, TRAPPIII, showed the Trs85 subunit is located at the end of the elongated TRAPPIII structure [29]. The positioning of Trs85 leaves Bet3 accessible for Sec23 binding suggesting that, like TRAPPI, TRAPPIII recruits Ypt1 to COPII vesicles [29]. This finding is consistent with a growing body of work implicating COPII vesicles in autophagosome formation.
COPII vesicles in autophagosome formation Although our understanding of the assembly of Atg proteins at the PAS has advanced significantly over the last two decades, the membrane trafficking events leading to autophagosome formation remain unclear. In yeast, two classes of vesicles, Atg9 and COPII, have been implicated in this process. Atg9 vesicles are trafficked from the transGolgi to the PAS and are required just downstream of the Atg1-initiating complex [30]. Although Atg9 vesicles are essential for phagophore initiation, only a few Atg9 vesicles are needed, indicating other membranes are involved [30]. COPII coat subunits were reported early on to be required for autophagosome formation [31,32]. These studies postulated that general flux of traffic from the ER might be needed for macroautophagy rather than a direct involvement of COPII vesicles [31]. Recent work, however, has begun to favour a model in which COPII vesicles directly contribute to autophagosome biogenesis [21,22,29]. The observation that the PAS is formed adjacent to ER-exit sites is suggestive of a role of COPII vesicles in autophagosome formation [24,25]. Consistent with this notion, when macroautophagy is blocked, COPII vesicles begin to accumulate at the PAS [29]. A recent proteomic study identified physical interactions between COPII coat subunits and many Atg proteins. The study by Graef et al. [24] went on to demonstrate COPII vesicles are required just downstream of the Atg1 initiating complex; however, the exact function of COPII vesicles at this step has yet to be determined. Moreover, many regulators of ER to Golgi trafficking, including Sar1 and the ER–Golgi SNAREs, were shown to be required for macroautophagy C The
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[29]. Notably, the only exception was the tether Uso1 [29]. This observation supports the notion that COPII vesicles are not indirectly playing a role in macroautophagy by transporting cargo to the Golgi. Furthermore, it raises the model that COPII vesicles are transported by a similar mechanism during macroautophagy, except a different tether targets COPII vesicles to a different acceptor compartment. The serine/threonine kinase Atg1 is the likely tether. Atg1 can directly bind membranes in vitro, which is consistent with reports that Atg1 has kinase-independent functions required upstream of Atg1 kinase activity [33,34]. Atg1 also directly binds to and phosphorylates Atg9 making it tempting to speculate Atg1, may tether Atg9 and COPII vesicles, although this model awaits further validation [35] (Figure 2).
Rab1 and mTRAPPIII in macroautophagy Although our understanding of the hierarchy of Atg proteins in the mammalian cells is less clear, these events appear to be generally conserved. In the mammalian cells, there is not just a single PAS, but multiple sites at which autophagosome formation is initiated. These sites have been localized to the ER and more specifically to the ER-exit sites [24,29]. Autophagosome formation in mammalian cells probably also involves Atg9 and COPII vesicles [36], although additional membrane sources, including ER–mitochondria contact sites, the mitochondria, the ER–Golgi intermediate compartment (ERGIC) and the plasma membrane, have also been implicated [37–40]. Rab1b and mammalian TRAPPIII are also required for autophagosome biogenesis [41,42]. Furthermore, Rab1 was reported to interact in vivo with Ulk1, the mammalian homologue of Atg1 [10]. Ulk1 forms a complex with the Atg17-like protein FIP200 [43,44]. Further work will be needed to verify whether FIP200 plays a similar role in recruiting mammalian TRAPPIII and Rab1 to sites of autophagosome formation.
Ypt1 and TRAPPIII during selective autophagy In addition to bulk macroautophagy, there exist several forms of selective autophagy that transport specific cargo, such as damaged organelles, for vacuolar degradation. Selective autophagy shares some of the same machinery as macroautophagy; however, as the pathways become better understood, clear differences have emerged. In yeast, the most well-studied form of selective autophagy is the cytoplasmic to vacuolar targeting (Cvt) pathway through which various vacuolar hydrolases are transported to the vacuole for activation. Ypt1 and TRAPPIII are both required for the Cvt pathway and may perform distinct functions from their roles during macroautophagy [11,45]. In nutrient-rich conditions, Ypt1 was reported to have a different effector, the Cvt-specific scaffold Atg11 [46]. Atg11 acts upstream in recruiting Atg proteins to the PAS for selective autophagy. Although Atg9 vesicles are required for both selective and macroautophagy
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Figure 2 Model of early autophagosome formation Upon induction of macroautophagy, the PAS is formed adjacent to the ER-exit sites and the vacuole. At the PAS, the Atg17 complex recruits TRAPPIII. TRAPPIII binds to the Sec23 subunit of the COPII coat and recruits and activates Ypt1. Activated Ypt1 aids in recruitment of Atg1. Atg1 binds and phosphorylates Atg9 and may tether Atg9 and COPII vesicles, initiating autophagosome formation.
[47], COPII vesicles do not seem to be required for the Cvt pathway [31]. TRAPPIII has been implicated in the transport of Atg9 vesicles from the endosome to the Golgi and this function is more essential in nutrient-rich conditions [48]. Ypt1 and TRAPPIII are probably involved in other forms of selective autophagy as Trs85 is required for the selective autophagy of peroxisomes and Rab1 and mammalian TRAPPIII were found to be required for antimicrobial autophagy [45,49]. Additional work will be needed to determine the role of Ypt1 during these other types of selective autophagy.
Concluding remarks The formation of the autophagosome has received significant attention in recent years resulting in a surge of new findings on this previously mysterious process. Work on the structure and function of Ypt1 and TRAPPIII have highlighted commonalities between the ER to Golgi pathway and the early stages of autophagosome biogenesis, namely the involvement of COPII vesicles. Although we have a good grasp of how directionality is achieved on the ER to Golgi pathway, our understanding of how COPII vesicles are redirected to the PAS to aid in autophagosome formation is unknown. As the tether Uso1 is not required for macroautophagy, tethering is probably an important decision point for the fate of COPII vesicles. It is also possible that the ER to Golgi trafficking machinery is modified during macroautophagy to preferentially interact with Atg proteins or to inhibit interactions with secretory machinery. Finally, COPII vesicles may be passively redirected to the PAS as the PAS is formed adjacent to ER-exit sites. This may allow the macroautophagy pathway to outcompete the secretory pathway for access to COPII vesicles. Understanding how the PAS is formed at ER-exit sites as well as obtaining a
better grasp of vesicle tethering and fusion events during phagophore initiation will be required in order to address these key questions.
Acknowledgements Salary support was provided by the Howard Hughes Medical Institute.
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Received 17 September 2014 doi:10.1042/BST20140247
