A Role for Macro-ER-Phagy in ER Quality Control

The endoplasmic-reticulum quality-control (ERQC) system shuttles misfolded proteins for degradation by the proteasome through the well-defined ER-associated degradation (ERAD) pathway. In contrast, very little is known about the role of autophagy in ERQC. Macro-autophagy, a collection of pathways that deliver proteins through autophagosomes (APs) for degradation in the lysosome (vacuole in yeast), is mediated by autophagy-specific proteins, Atgs, and regulated by Ypt/Rab GTPases. Until recently, the term ER-phagy was used to describe degradation of ER membrane and proteins in the lysosome under stress: either ER stress induced by drugs or whole-cell stress induced by starvation. These two types of stresses induce micro-ER-phagy, which does not use autophagic organelles and machinery, and non-selective autophagy. Here, we characterize the macro-ER-phagy pathway and uncover its role in ERQC. This pathway delivers 20–50% of certain ER-resident membrane proteins to the vacuole and is further induced to >90% by overexpression of a single integral-membrane protein. Even though such overexpression in cells defective in macro-ER-phagy induces the unfolded-protein response (UPR), UPR is not needed for macro-ER-phagy. We show that macro-ER-phagy is dependent on Atgs and Ypt GTPases and its cargo passes through APs. Moreover, for the first time the role of Atg9, the only integral-membrane core Atg, is uncoupled from that of other core Atgs. Finally, three sequential steps of this pathway are delineated: Atg9-dependent exit from the ER en route to autophagy, Ypt1- and core Atgs-mediated pre-autophagsomal-structure organization, and Ypt51-mediated delivery of APs to the vacuole.     

A Ypt/Rab GTPase module makes a PAS

Organization of membrane micro-domains by Ypt/Rab GTPases is key for all membrane trafficking events in eukaryotic cells. Since autophagy is a membrane trafficking process, it was expected that these GTPases would play a role in autophagy as well. While evidence about participation of Ypt/Rabs in autophagy is beginning to emerge, the mechanisms by which they act in this process are still not clear. Moreover, it is still questionable if and how Ypt/Rabs coordinate autophagy with other cellular trafficking processes. Yeast Ypt1 and its mammalian homolog Rab1 are required for both endoplasmic reticulum (ER)-to-Golgi transport and autophagy, suggesting that they coordinate these two processes. In our recent paper, we identify Atg11, a bona fide phagophore assembly site (PAS) component, as a downstream effector of Ypt1. Moreover, we show that three components of a GTPase module—the Ypt1 activator, Trs85-containing TRAPP complex, Ypt1, and the Atg11 effector—interact on the PAS and are required for PAS formation during selective autophagy. We propose that Ypt/Rabs coordinate the secretory and the autophagic pathways by recruiting process-specific effectors.     

Regulation of ER-phagy by a Ypt/Rab GTPase module

Accumulation of misfolded proteins on intracellular membranes has been implicated in neurodegenerative diseases. One cellular pathway that clears such aggregates is endoplasmic reticulum autophagy (ER-phagy), a selective autophagy pathway that delivers excess ER to the lysosome for degradation. Not much is known about the regulation of ER-phagy. The conserved Ypt/Rab GTPases regulate all membrane trafficking events in eukaryotic cells. We recently showed that a Ypt module, consisting of Ypt1 and autophagy-specific upstream activator and downstream effector, regulates the onset of selective autophagy in yeast. Here we show that this module acts at the ER. Autophagy-specific mutations in its components cause accumulation of excess membrane proteins on aberrant ER structures and induction of ER stress. This accumulation is due to a block in transport of these membranes to the lysosome, where they are normally cleared. These findings establish a role for an autophagy-specific Ypt1 module in the regulation of ER-phagy. Moreover, because Ypt1 is a known key regulator of ER-to-Golgi transport, these findings establish a second role for Ypt1 at the ER. We therefore propose that individual Ypt/Rabs, in the context of distinct modules, can coordinate alternative trafficking steps from one cellular compartment to different destinations.     

The GTPase-activating enzyme Gyp1p is required for recycling of internalized membrane material by inactivation of the Rab/Ypt GTPase Ypt1p

Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1 Delta cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.     

A Ypt32p exchange factor is a putative effector of Ypt1p

Ypt1p regulates vesicle tethering and fusion events from the ER to the Golgi and through the early Golgi. Genetic studies have suggested a functional relationship between Ypt1p and Ypt31p/Ypt32p. Ypt31p and Ypt32p are a pair of functionally redundant GTPases that act after Ypt1p to mediate intra-Golgi traffic or the budding of post-Golgi vesicles from the trans-Golgi. Here we report that a novel Ypt32p exchange factor is a putative effector of Ypt1p. These findings implicate small GTP-binding proteins of the Ypt/Rab family in a signal cascade that directs membrane traffic through the secretory pathway.     

The Yip1p.Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles

Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p.Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.     

Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.

The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.     

An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes.

Membrane traffic requires vesicles to fuse with a specific target, and SNARE proteins and Rab/Ypt GTPases contribute to this specificity. In the yeast Saccharomyces cerevisae, the Rab/Ypt GTPase Ypt6p is required for fusion of endosome-derived vesicles with the late Golgi. We have shown previously that activation of Ypt6p depends on its exchange factor, Ric1p-Rgp1p, a peripheral membrane protein complex restricted to the Golgi. We show here that a conserved trimeric protein complex, VFT (Vps52/53/54), binds directly to Ypt6p:GTP. Localization of VFT to the Golgi requires Ypt6p, but is unaffected in gos1 and tlg1 mutants, in which late Golgi integral membrane proteins, including SNAREs, are mislocalized. The VFT complex also binds directly to the N-terminal domain of the SNARE Tlg1p, both in vitro and in vivo, in a Ypt6p-independent manner. We suggest that the VFT complex links vesicles containing Tlg1p to their target, which is defined by the local activation of Ypt6p.     

Identification of Discrete Sites in Yip1A Necessary for Regulation of Endoplasmic Reticulum Structure

The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.     

Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.     

GDP dissociation inhibitor domain II required for Rab GTPase recycling

Rab GTPases are localized to distinct subsets of organelles within the cell, where they regulate SNARE-mediated membrane trafficking between organelles. One factor required for Rab localization and function is Rab GDP dissociation inhibitor (GDI), which is proposed to recycle Rab after vesicle fusion by extracting Rab from the membrane and loading Rab onto newly formed transport intermediates. GDI is composed of two domains; Rab binding is mediated by Domain I, and the function of Domain II is not known. In this study, Domain II of yeast GDI, encoded by the essential GDI1/SEC19 gene, was targeted in a genetic screen to obtain mutants that might lend insight into the function of this domain. In one gdi1 mutant, the cytosolic pools of all Rabs tested were depleted, and Rab accumulated on membranes, suggesting that this mutant Gdi1 protein has a general defect in extraction of Rab from membranes. In a second gdi1 mutant, the endosomal/vacuolar Rabs Vps21/Ypt51p and Ypt7p accumulated in the cytosol bound to Gdi1p, but localization of Ypt1p and Sec4p were not significantly affected. Using an in vitro assay which reconstitutes Gdi1p-mediated membrane loading of Rab, this mutant Gdi1p was found to be defective in loading of Vps21p but not Ypt1p. Loading of Vps21p by loading-defective Gdi1p was restored when acceptor membranes prepared from a deletion strain lacking Vps21p were used. These results suggest that membrane-associated Rab may regulate recruitment of GDI-Rab from the cytosol, possibly by regulating a GDI-Rab receptor. We conclude that Domain II of Gdi1p is essential for Rab loading and Rab extraction, and confirm that each of these activities is required for Gdi1p function in vivo.     

The HOPS/Class C Vps Complex Tethers High‐Curvature Membranes via a Direct Protein–Membrane Interaction

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter‐membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low‐curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p‐bearing low‐curvature liposomes even when the high‐curvature liposomes are protein‐free. Phosphorylation of the curvature‐sensing amphipathic lipid‐packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high‐curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high‐curvature liposomes and Ypt7p‐bearing low‐curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein–membrane interaction. Such high‐curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole‐vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high‐curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane.      

Arf1 orchestrates Rab GTPase conversion at the trans-Golgi network

Rab family GTPases are key organizers of membrane trafficking and function as markers of organelle identity. Accordingly, Rab GTPases often occupy specific membrane domains, and mechanisms exist to prevent the inappropriate mixing of distinct Rab domains. The yeast Golgi complex can be divided into two broad Rab domains: Ypt1 (Rab1) and Ypt6 (Rab6) are present at the early/medial Golgi and sharply transition to Ypt31/32 (Rab11) at the late Golgi/trans-Golgi network (TGN). This Rab conversion has been attributed to GTPase-activating protein (GAP) cascades in which Ypt31/32 recruits the Rab-GAPs Gyp1 and Gyp6 to inactivate Ypt1 and Ypt6, respectively. Here we report that Rab transition at the TGN involves additional layers of regulation. We provide new evidence confirming the TRAPPII complex as an important regulator of Ypt6 inactivation and uncover an unexpected role of the Arf1 GTPase in recruiting Gyp1 to drive Ypt1 inactivation at the TGN. Given its established role in directly recruiting TRAPPII to the TGN, Arf1 is therefore a master regulator of Rab conversion on maturing Golgi compartments.     

Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole

Transport within the endocytic pathway depends on a consecutive function of the endosomal Rab5 and the late endosomal/lysosomal Rab7 GTPases to promote membrane recycling and fusion in the context of endosomal maturation. We previously identified the hexameric BLOC-1 complex as an effector of the yeast Rab5 Vps21, which also recruits the GTPase-activating protein (GAP) Msb3. This raises the question of when Vps21 is inactivated on endosomes. We provide evidence for a Rab cascade in which activation of the Rab7 homologue Ypt7 triggers inactivation of Vps21. We find that the guanine nucleotide exchange factor (GEF) of Ypt7 (the Mon1-Ccz1 complex) and BLOC-1 both localize to the same endosomes. Overexpression of Mon1-Ccz1, which generates additional Ypt7-GTP, or overexpression of activated Ypt7 promotes relocalization of Vps21 from endosomes to the endoplasmic reticulum (ER), which is indicative of Vps21 inactivation. This ER relocalization is prevented by loss of either BLOC-1 or Msb3, but it also occurs in mutants lacking endosome–vacuole fusion machinery such as the HOPS tethering complex, an effector of Ypt7. Importantly, BLOC-1 interacts with the HOPS on vacuoles, suggesting a direct Ypt7-dependent cross-talk. These data indicate that efficient Vps21 recycling requires both Ypt7 and endosome–vacuole fusion, thus suggesting extended control of a GAP cascade beyond Rab interactions.     

Interaction of the Saccharomyces cerevisiae cortical actin patch protein Rvs167p with proteins involved in ER to Golgi vesicle trafficking

We have used affinity chromatography to identify two proteins that bind to the SH3 domain of the actin cytoskeleton protein Rvs167p: Gyp5p and Gyl1p. Gyp5p has been shown to be a GTPase activating protein (GAP) for Ypt1p, a Rab GTPase involved in ER to Golgi trafficking; Gyl1p is a protein that resembles Gyp5p and has recently been shown to colocalize with and belong to the same protein complex as Gyp5p. We show that Gyl1p and Gyp5p interact directly with each other, likely through their carboxy-terminal coiled-coil regions. In assays of GAP activity, Gyp5p had GAP activity toward Ypt1p and we found that this activity was stimulated by the addition of Gyl1p. Gyl1p had no GAP activity toward Ypt1p. Genetic experiments suggest a role for Gyp5p and Gyl1p in ER to Golgi trafficking, consistent with their biochemical role. Since Rvs167p has a previously characterized role in endocytosis and we have shown here that it interacts with proteins involved in Golgi vesicle trafficking, we suggest that Rvs167p may have a general role in vesicle trafficking.     

The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex

A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)-Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3'-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity--that is, beyond bulk effector recruitment--for a Rab GTPase.     

A Rab requirement is not bypassed in SLY1-20 suppression

The Rab GTPase Ypt1p and the large homodimer Uso1p are both required for tethering endoplasmic reticulum-derived vesicles to early Golgi compartments in yeast. Loss-of-function ypt1 and uso1 mutations are suppressed by SLY1-20, a dominant allele that encodes the Sed5p-associated protein, Sly1p. Here, we investigate the mechanism of SLY1-20 suppression. In wild-type strains, Ypt1p can be coimmunoprecipitated with Uso1p; however, in a ypt1Delta/SLY1-20 strain, which lacks this complex, membrane binding of Uso1p was reduced. In spite of Ypt1p depletion, Uso1p-dependent vesicle tethering was not bypassed under the ypt1Delta/SLY1-20 condition. Moreover, tethering and fusion assays with ypt1Delta/SLY1-20 membranes remained sensitive to Rab GDP dissociation inhibitor. These results indicate that an alternative Rab protein satisfies the Ypt1p requirement in Uso1p-dependent tethering when SLY1-20 is expressed. Further genetic and biochemical tests revealed that a related Rab protein, Ypt6, might substitute for Ypt1p in ypt1Delta/SLY1-20 cells. Additional experimentation to address the mechanism of SLY1-20 suppression in a cog2Delta [sec35Delta] strain indicated that the Cog2p subunit of the conserved oligomeric Golgi complex is either functionally redundant or is not directly required for anterograde transport to the Golgi complex.