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The target of rapamycin complex 2 (TORC2) signaling is associated with plasma membrane (PM) integrity. In Saccharomyces cerevisiae, TORC2-Ypk1/2 signaling controls sphingolipid biosynthesis, and Ypk1/2 phosphorylation by TORC2 under PM stress conditions is increased in a Slm1/2-dependent manner, under which Slm1 is known to be released from an eisosome, a furrow-like invagination PM structure. However, it remains unsolved how the activation machinery of TORC2-Ypk1/2 signaling is regulated. Here we show that edelfosine, a synthetic lysophospholipid analog, inhibits the activation of TORC2-Ypk1/2 signaling, and the cell wall integrity (CWI) pathway is involved in this inhibitory effect. The activation of CWI pathway blocked the eisosome disassembly promoted by PM stress and the release of Slm1 from eisosomes. Constitutive activation of TORC2-Ypk1/2 signaling exhibited increased sensitivity to cell wall stress. We propose that the CWI pathway negatively regulates the TORC2-Ypk1/2 signaling, which is involved in the regulatory mechanism to ensure the proper stress response to cell wall damage., (© 2024. The Author(s).)

Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function., (© 2024 Füllbrunn et al.)

The autophagy initiation complex is brought about via a highly ordered and stepwise assembly process. Two crucial signaling molecules, mTORC1 and AMPK, orchestrate this assembly by phosphorylating/dephosphorylating autophagy-related proteins. Activation of Atg1 followed by recruitment of both Atg9 vesicles and the PI3K complex I to the PAS (phagophore assembly site) are particularly crucial steps in its formation. Ypt1, a small Rab GTPase in yeast cells, also plays an essential role in the formation of the autophagy initiation complex through multiple regulatory pathways. In this review, our primary focus is to discuss how signaling molecules initiate the assembly of the autophagy initiation complex, and highlight the significant roles of Ypt1 in this process. We end by addressing issues that need future clarification., (© 2024 Wiley Periodicals LLC.)

Tethering factors play a critical role in deciphering the correct combination of vesicle and target membrane, before SNARE complex formation and membrane fusion. The exocyst plays a central role in tethering post-Golgi vesicles to the plasma membrane, although the mechanism by which this occurs is poorly understood. We recently established an assay for measuring exocyst-mediated vesicle tethering in vitro and we have adapted this assay to examine the ability of exocyst to tether vesicles in an asymmetric manner. We demonstrate that exocyst differs from another post-Golgi vesicle tethering protein, Sro7, in that it is fully capable of tethering vesicles with a functional Rab GTPase, Sec4, to vesicles lacking a functional Rab GTPase. Using this assay, we show that exocyst requires both the Rab and R-SNARE, Snc1, to be present on the same membrane surface. Using Sac1 phosphatase treatment, we demonstrate a likely role for phosphoinositides on the opposing Rab-deficient membrane. This suggests a specific model for exocyst orientation and its points of contact between membranes during heterotypic tethering of post-Golgi vesicles with the plasma membrane.

Reprogramming organelle size has been proposed as a potential therapeutic approach. However, there have been few reports of nucleolar size reprogramming. We addressed this question in Saccharomyces cerevisiae by studying mutants having opposite effects on the nucleolar size. Mutations in genes involved in nuclear functions (KAR3, CIN8, and PRP45) led to enlarged nuclei/nucleoli, whereas mutations in secretory pathway family genes, namely the Rab-GTPases YPT6 and YPT32, reduced nucleolar size. When combined with mutations leading to enlarged nuclei/nucleoli, the YPT6 or YPT32 mutants can effectively reprogram the nuclear/nucleolar size almost back to normal. Our results further indicate that null mutation of YPT6 causes secretory stress that indirectly influences nuclear localization of Maf1, the negative regulator of RNA Polymerase III, which might reduce the nucleolar size by inhibiting nucleolar transcript enrichment., (© 2023 Federation of European Biochemical Societies.)

Polarized secretion facilitates polarized cell growth. For a secretory vesicle to dock at the plasma membrane, it must mature with a progressive association or dissociation of molecules that are, respectively, necessary for or inhibitory to vesicle docking, including an exchange of Rab GTPases. In current models, oxysterol-binding protein homologue 4 (Osh4p) establishes a phosphatidylinositol 4-phosphate (PI4P) gradient along the secretory trafficking pathway such that vesicles have higher PI4P levels after budding from the trans -Golgi relative to when vesicles arrive at the plasma membrane. In this study, using the lipid-binding domain P4M and live-cell imaging, we show that secretory vesicle-associated PI4P levels remain constant when vesicles traffic from the trans -Golgi to the plasma membrane. We also show that deletion of OSH4 does not alter vesicle-associated PI4P levels, though loss of any individual member of the OSH family or complete loss of OSH family function alters the intracellular distribution of PI4P. We propose a model in which the Rab GTPases Ypt32p and Sec4p remain associated with a secretory vesicle during trafficking, independent of PI4P levels and Osh4p. Together these data indicate the necessity of experiments revealing the location and timing of events required for vesicle maturation.

Clathrin-mediated vesicle trafficking plays central roles in post-Golgi transport. In yeast (Saccharomyces cerevisiae), the AP-1 complex and GGA adaptors are predicted to generate distinct transport vesicles at the trans-Golgi network (TGN), and the epsin-related proteins Ent3p and Ent5p (collectively Ent3p/5p) act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGA adaptors are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here, we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We show that AP-1 disruption in the ent3Δ5Δ mutant impaired transport of the Vps21p guanine nucleotide exchange factor Vps9p transport to the Vps21p compartment and severely reduced Vps21p activity. Additionally, GGA adaptors, the phosphatidylinositol-4-kinase Pik1p and Rab11 GTPases Ypt31p and Ypt32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.

The Saccharomyces cerevisiae casein kinase protein Yck3 is a central regulator at the vacuole that phosphorylates several proteins involved in membrane trafficking. Here, we set out to identify novel substrates of this protein. We found that endogenously tagged Yck3 localized not only at the vacuole, but also on endosomes. To disable Yck3 function, we generated a kinase-deficient mutant and thus identified the I-BAR-protein Ivy1 as a novel Yck3 substrate. Ivy1 localized to both endosomes and vacuoles, and Yck3 controlled this localization. A phosphomimetic Ivy1-SD mutant was found primarily on vacuoles, whereas its non-phosphorylatable SA variant strongly localized to endosomes, similar to what was observed upon deletion of Yck3. In vitro analysis revealed that Yck3-mediated phosphorylation strongly promoted Ivy1 recruitment to liposomes carrying the Rab7-like protein Ypt7. Modeling of Ivy1 with Ypt7 identified binding sites for Ypt7 and a positively charged patch, which were both required for Ivy1 localization. Strikingly, Ivy1 mutations in either site resulted in more cells with multilobed vacuoles, suggesting a partial defect in its membrane biogenesis. Our data thus indicate that Yck3-mediated phosphorylation controls both localization and function of Ivy1 in endolysosomal biogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The exocyst imparts spatial control during exocytic vesicle tethering through its interactions with proteins and lipids on the vesicle and the plasma membrane. One such interaction is with the vesicle tether Sro7, although the outcome of this interaction is poorly understood. Here, we describe how Sro7 binding to the Exo84 subunit results in activation of the exocyst complex which leads to an increase in avidity for the Rab GTPase Sec4 and an increase in exocyst-mediated vesicle tethering. Gain-of-function (GOF) mutations in Exo84 that mimic Sro7 activation replicate these biochemical changes and result in allosteric changes within the complex. Direct comparison of GOF mutants which mimic Sro7- and Rho/Cdc42-activation of the exocyst reveals distinct mechanisms and outcomes. We propose a model by which these two activation pathways reside within the same tethering complex but remain insulated from one another. Structural modeling suggests a related mechanism for Sro7 activation of the exocyst in yeast and Ral GTPase activation of the exocyst in animal cells., (© 2023 Miller et al.)

Correct localization of Rab GTPases in cells is critical for proper function in membrane trafficking. Guanine-nucleotide exchange factors (GEFs) act as the primary determinants of Rab localization by activating and stabilizing their Rab substrates on specific organelle and vesicle membranes. The TRAPP complexes TRAPPII and TRAPPIII are two related GEFs that use the same catalytic site to activate distinct Rabs, Rab11 and Rab1, respectively. The Rab C-terminal hypervariable domain (HVD) is an important specificity determinant for the budding yeast TRAPP complexes, with the length of the HVD playing a critical role in counter-selection. Several recent studies have used cryo-EM to illuminate how the yeast and metazoan TRAPP complexes identify and activate their substrates. This review summarizes recently characterized Rab substrate selection mechanisms and highlights how the membrane surface provides critical context for the GEF-GTPase interactions., (© 2022 Federation of European Biochemical Societies.)

Induction of unfolded protein response involves activation of transcription factor Hac1p that is encoded by HAC1 pre-mRNA harboring an intron and a bipartite element (BE), which is subjected to nuclear mRNA decay by the nuclear exosome/Cbc1p-Tif4631p-dependent Exosome Targeting (CTEXT) complex. Using a combination of genetic and biochemical approaches, we demonstrate that a Rab-GTPase Ypt1p controls unfolded protein response signaling dynamics. This regulation relies on the nuclear localization of a small fraction of the cellular Ypt1p pool in the absence of endoplasmic reticulum (ER)-stress causing a strong association of the nuclear Ypt1p with pre- HAC1 mRNA that eventually promotes sequential recruitments of NNS, CTEXT, and the nuclear exosome onto this pre-mRNA. Recruitment of these decay factors onto pre- HAC1 mRNA is accompanied by its rapid nuclear decay that produces a precursor RNA pool lacking functional BE thereby causing its inefficient targeting to Ire1p foci leading to their diminished splicing and translation. ER stress triggers rapid relocalization of the nuclear pool of Ypt1p to the cytoplasm leading to its dissociation from pre- HAC1 mRNA thereby causing decreased recruitment of these decay factors to precursor HAC1 RNA leading to its diminished degradation. Reduced decay results in an increased abundance of pre- HAC1 mRNA with intact functional BE leading to its enhanced recruitment to Ire1p foci.

The mating of budding yeast depends on chemotropism, a fundamental cellular process. Haploid yeast cells of opposite mating type signal their positions to one another through mating pheromones. We have proposed a deterministic gradient sensing model that explains how these cells orient toward their mating partners. Using the cell-cycle determined default polarity site (DS), cells assemble a gradient tracking machine (GTM) composed of signaling, polarity, and trafficking proteins. After assembly, the GTM redistributes up the gradient, aligns with the pheromone source, and triggers polarized growth toward the partner. Since positive feedback mechanisms drive polarized growth at the DS, it is unclear how the GTM is released for tracking. What prevents the GTM from triggering polarized growth at the DS? Here, we describe two mechanisms that are essential for tracking: inactivation of the Ras GTPase Bud1 and positioning of actin-independent vesicle delivery upgradient., (© 2022 Wang et al.)

Most of the components in the yeast secretory pathway have been studied, yet a high-resolution temporal timeline of their participation is lacking. Here, we define the order of acquisition, lifetime, and release of critical components involved in late secretion from the Golgi to the plasma membrane. Of particular interest is the timing of the many reported effectors of the secretory vesicle Rab protein Sec4, including the myosin-V Myo2, the exocyst complex, the lgl homolog Sro7, and the small yeast-specific protein Mso1. At the trans-Golgi network (TGN) Sec4's GEF, Sec2, is recruited to Ypt31-positive compartments, quickly followed by Sec4 and Myo2 and vesicle formation. While transported to the bud tip, the entire exocyst complex, including Sec3, is assembled on to the vesicle. Before fusion, vesicles tether for 5 s, during which the vesicle retains the exocyst complex and stimulates lateral recruitment of Rho3 on the plasma membrane. Sec2 and Myo2 are rapidly lost, followed by recruitment of cytosolic Sro7, and finally the SM protein Sec1, which appears for just 2 s prior to fusion. Perturbation experiments reveal an ordered and robust series of events during tethering that provide insights into the function of Sec4 and effector exchange., Competing Interests: RG, AS, JP, AB No competing interests declared, (© 2022, Gingras et al.)

It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy., Competing Interests: The authors have declared that no competing interests exist.

Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and pathogenic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requires HOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPS binds small membrane-associated GTPases and assembles SNAREs for fusion, but how the complex fulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the structure of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities, where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore, ideally positioned between the membranes to catalyze fusion. Our data suggest a model for how HOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part of the membrane fusion machinery., Competing Interests: DS, JS, CK, AP, NF, SK, LL, DJ, KS, DK, FF, AM, CU No competing interests declared, (© 2022, Shvarev, Schoppe, König et al.)

Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21 ∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21 ∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy.

Two papers in this issue provide new structural insights into the "TRAnsport Protein Particle" (TRAPP) complexes, which play crucial roles in Golgi function. Both papers focus on TRAPPIII, which activates the Rab protein Ypt1 in yeast or the homologous Rab1 in metazoans. The structures illuminate how TRAPPIII specifically recognizes its Rab protein substrate. Joiner et al (2021) also describe a membrane-anchoring mechanism for yeast TRAPPIII, while Galindo et al (2021) characterize the large subunits that define metazoan TRAPPIII., (© 2021 The Authors.)

The yeast vacuole is functionally corresponding to vacuoles in eukaryote cells, it consists of a fusion protein that assists in the fusion of vacuoles and plays an important role in many processes. In addition, chemicals such as NH 4 Cl can reduce the size of vacuoles but as a side effect that also inhibits vacuoles making them inactive. In this study, to develop pre-treatments for extending the life of cut flowers, we constructed recombinant yeast using the fusion protein YPT7 and confirmed the activity of down-sized vacuoles. All the vacuoles of the recombinant yeast except vacuoles from recombinant yeast (MBTL-MYH-3) were found to be small vacuoles than mock (MBTL-MYH-0) and YPT7 overexpression model (MBTL-MYH-1). To confirm their activity, we conducted a test for antimicrobial activity. The results showed the other vacuoles of recombinant yeast had lower antimicrobial activity than the mock control, most of them showed about 60 % to 80 % of the antimicrobial activity. However, MBTL-MYH-3, whose vacuole did not change its size, showed antimicrobial activity lower than 40 %. Therefore, the cut flowers are better able to absorb smaller vacuoles after using the fusion protein YPT7. We expect that absorbing vacuoles more effective to senescence of cut flower than vacuolar enzymes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Membrane tethers play a critical role in organizing the complex molecular architecture of eukaryotic cells. Uso1 (yeast homolog of human p115) is essential for tethering in vesicle transport from ER to Golgi and interacts with Ypt1 GTPase. The N-terminal globular head domain of Uso1 is responsible for Ypt1 binding; however, the mechanism of tethering between ER transport vesicles and Golgi is unknown. Here, we determined two crystal structures for the Uso1 N-terminal head domain in two alternative conformations. The head domain of Uso1 exists as a monomer, as confirmed using size-exclusion chromatography coupled to multi-angle light scattering and analytical gel filtration. Although Uso1 consists of a right-handed α-solenoid, like that in mammalian homologs, the overall conformations of both Uso1 structures were not similar to previously known p115 structures, suggesting that it adopts alternative conformations. We found that the N- and C-terminal regions of the Uso1 head domain are connected by a long flexible linker, which may mediate conformational changes. To analyse the role of the alternative conformations of Uso1, we performed molecular docking of Uso1 with Ypt1, followed by a structural comparison. Taken together, we hypothesize that the alternative conformations of Uso1 regulate the precise docking of vesicles to Golgi.

Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine nucleotide exchange factor (GEF), which is Mon1-Ccz1 for Rab7. During endosome maturation, Rab5 is replaced by Rab7, though the underlying mechanism remains poorly understood. Here, we identify the molecular determinants for Rab conversion in vivo and in vitro, and reconstitute Rab7 activation with yeast and metazoan proteins. We show (i) that Mon1-Ccz1 is an effector of Rab5, (ii) that membrane-bound Rab5 is the key factor to directly promote Mon1-Ccz1 dependent Rab7 activation and Rab7-dependent membrane fusion, and (iii) that this process is regulated in yeast by the casein kinase Yck3, which phosphorylates Mon1 and blocks Rab5 binding. Our study thus uncovers the minimal feed-forward machinery of the endosomal Rab cascade and a novel regulatory mechanism controlling this pathway., Competing Interests: LL, AB, EH, NF, YH, AP, KA, DK, CU No competing interests declared, (© 2020, Langemeyer et al.)

Intracellular membrane fusion requires Rab-family GTPases, their effector tethers, soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, and SNARE chaperones of the Sec1/Munc18 (SM), Sec17/α-SNAP, and Sec18/NSF families. We have developed an assay using fluorescence resonance energy transfer to measure SNARE complex formation in real time. We now show that yeast vacuolar SNAREs assemble spontaneously into RQaQbQc complexes when the R- and Qa-SNAREs are concentrated in the same micelles or in cis on the same membrane. When SNAREs are free in solution or are tethered to distinct membranes, assembly requires catalysis by HOPS, the vacuolar SM and tethering complex. The Rab Ypt7 and vacuole lipids together allosterically activate the bound HOPS for catalyzing SNARE assembly, even if none of the SNAREs are membrane bound. HOPS-dependent fusion between proteoliposomes bearing R- or Qa-SNAREs shows a strict requirement for Ypt7 on the R-SNARE proteoliposomes but not on the Qa-SNARE proteoliposomes. This asymmetry is reflected in the strikingly different capacity of Ypt7 in cis to either the R- or Qa-SNARE to stimulate SNARE complex assembly. Membrane-bound Ypt7 activates HOPS to catalyze 4-SNARE complex assembly when it is on the same membrane as the R-SNARE but not the Qa-SNARE, thus explaining the asymmetric need for Ypt7 for fusion.

The Cdc42 guanosine triphosphatase (GTPase) plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site is selected in the G1 phase. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet how Rsr1 regulates cell polarization is not fully understood. Here, we show that Rsr1-GDP interacts with the scaffold protein Bem1 in early G1, likely hindering the role of Bem1 in Cdc42 polarization and polarized secretion. Consistent with these in vivo observations, mathematical modeling predicts that Bem1 is unable to promote Cdc42 polarization in early G1 in the presence of Rsr1-GDP. We find that a part of the Bem1 Phox homology domain, which overlaps with a region interacting with the exocyst component Exo70, is necessary for the association of Bem1 with Rsr1-GDP. Overexpression of the GDP-locked Rsr1 interferes with Bem1-dependent Exo70 polarization. We thus propose that Rsr1 functions in spatial and temporal regulation of polarity establishment by associating with distinct polarity factors in its GTP- and GDP-bound states.

Autophagy is an intracellular degradation process involving many Atg proteins, which are recruited hierarchically to regulate this process. Rab/Ypt GTPases and their activators, guanine nucleotide exchange factors (GEFs), which are critical for regulating vesicle trafficking, are also involved in autophagy. Previously, we reported that yeast Vps21 and its GEF Vps9 are required for autophagy. Later, a third yeast VPS9-domain-containing protein, VARP-like 1 (Vrl1), which was identified as a mutant in major laboratory strains, had partially overlapping functions with Vps9 in trafficking. In this study, we showed that Vrl1 performed roles in autophagy, and its VPS9-domain was crucial for its role in autophagy. We found that localization of Vrl1 differed from the other two VPS9-domain-containing proteins, Vps9 and Muk1, and only Vrl1 changed from multipoint to diffusion after starvation. Like Vps9, Vrl1 suppressed autophagic defects caused by the VPS9 deletion. We further showed that these VPS9-domain-containing proteins, Vps9, Muk1, and Vrl1, all co-localized with Atg8 on autophagosomes in cells blocked in any late step of starvation-induced autophagy, with Vrl1 most often co-localizing with Atg8. A small portion (<25%) of these VPS9-domain-containing proteins were degraded through autophagy. However, a large portion (>60%) of Vrl1 decreased independently of autophagy. We propose that Vrl1 may regulate autophagy in a similar way as Vps9, and the level of Vrl1 partly decreases through both autophagy-dependent and -independent routes., (© 2019 International Federation for Cell Biology.)

Macroautophagy/autophagy is a highly conserved intracellular vesicle transport pathway that prevents accumulation of harmful materials within cells. The dynamic assembly and disassembly of the different autophagic protein complexes at the so-called phagophore assembly site (PAS) is strictly regulated. Rab GTPases are major regulators of cellular vesicle trafficking, and the Rab GTPase Ypt1 and its GEF TRAPPIII have been implicated in autophagy. We show that Gyp1 acts as a Ypt1 GTPase-activating protein (GAP) for selective autophagic variants, such as the Cvt pathway or the selective autophagic degradation of mitochondria (mitophagy). Gyp1 regulates the dynamic disassembly of the conserved Ypt1-Atg1 complex. Thereby, Gyp1 sets the stage for efficient Atg14 recruitment, and facilitates the critical step from nucleation to elongation of the phagophore. In addition, we identified Gyp1 as a new Atg8-interacting motif (AIM)-dependent Atg8 interaction partner. The Gyp1 AIM is required for efficient formation of the cargo receptor-Atg8 complexes. Our findings elucidate the molecular mechanisms of complex disassembly during phagophore formation and suggest potential dual functions of GAPs in cellular vesicle trafficking. Abbreviations AIM, Atg8-interacting motif; Atg, autophagy related; Cvt, cytoplasm-to-vacuole targeting; GAP, GTPase-activating protein; GEF, guanine-nucleotide exchange factor; GFP, green fluorescent protein; log phase, logarithmic growth phase; NHD, N-terminal helical domain; PAS, phagophore assembly site; PE, phosphatidylethanolamine; PtdIns3P, phosphatidylinositol-3-phosphate; WT, wild-type.

The Arf and Rab/Ypt GTPases coordinately regulate membrane traffic and organelle structure by regulating vesicle formation and fusion. Ample evidence has indicated that proteins in these two families may function in parallel or complementarily; however, the manner in which Arf and Rab/Ypt proteins perform interchangeable functions remains unclear. In this study, we report that a Golgi-localized Arf, Arl1, could suppress Ypt6 dysfunction via its effector golgin, Imh1, but not via the lipid flippase Drs2. Ypt6 is critical for the retrograde transport of vesicles from endosomes to the trans-Golgi network (TGN), and its mutation leads to severe protein mislocalization and growth defects. We first overexpress the components of the Arl3-Syt1-Arl1-Imh1 cascade and show that only Arl1 and Imh1 can restore endosome-to-TGN trafficking in ypt6-deleted cells. Interestingly, increased abundance of Arl1 or Imh1 restores localization of the tethering factor Golgi associated retrograde-protein (GARP) complex to the TGN in the absence of Ypt6. We further show that the N-terminal domain of Imh1 is critical for restoring GARP localization and endosome-to-TGN transport in ypt6-deleted cells. Together, our results reveal the mechanism by which Arl1-Imh1 facilitates the recruitment of GARP to the TGN and compensates for the endosome-to-TGN trafficking defects in dysfunctional Ypt6 conditions.

Correct localization of Rab GTPases in cells is critical for proper function in membrane trafficking, yet the mechanisms that target Rabs to specific subcellular compartments remain controversial. Guanine nucleotide exchange factors (GEFs) activate and consequently stabilize Rab substrates on membranes, thus implicating GEFs as the primary determinants of Rab localization. A competing hypothesis is that the Rab C-terminal hypervariable domain (HVD) serves as a subcellular targeting signal. In this study, we present a unifying mechanism in which the HVD controls targeting of certain Rabs by mediating interaction with their GEFs. We demonstrate that the TRAPP complexes, two related GEFs that use the same catalytic site to activate distinct Rabs, distinguish between Ypt1 (Rab1) and Ypt31/32 (Rab11) via their divergent HVDs. Remarkably, we find that HVD length gates Rab access to the TRAPPII complex by constraining the distance between the nucleotide-binding domain and the membrane surface., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In our previous study, we found that Ypt1p, a Rab family small GTPase protein, exhibits a stress-driven structural and functional switch from a GTPase to a molecular chaperone, and mediates thermo tolerance in Saccharomyces cerevisiae . In the current study, we focused on the temperature-sensitive ypt1-G80D mutant, and found that the mutant cells are highly sensitive to heat-shock, due to a deficiency in the chaperone function of Ypt1p G80D . This defect results from an inability of the protein to form high molecular weight polymers, even though it retains almost normal GTPase function. The heat-stress sensitivity of ypt1-G80D cells was partially recovered by treatment with 4-phenylbutyric acid, a chemical chaperone. These findings indicate that loss of the chaperone function of Ypt1p G80D underlies the heat sensitivity of ypt1-G80D cells. We also compared the proteomes of YPT1 (wild-type) and ypt1-G80D cells to investigate Ypt1p-controlled proteins under heat-stress conditions. Our findings suggest that Ypt1p controls an abundance of proteins involved in metabolism, protein synthesis, cellular energy generation, stress response, and DNA regulation. Finally, we suggest that Ypt1p essentially regulates fundamental cellular processes under heat-stress conditions by acting as a molecular chaperone.

Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when the interaction between the Rab-GTPase Ypt7 and its effector Vps41 (a subunit of the tethering complex HOPS) is interrupted by a point mutation (Ypt7-D44N). Subsequent phosphorylation of Vps41 by the casein kinase Yck3 prevents stabilization of trans-SNARE complexes needed for lipid bilayer pore formation. Impairing ILF formation prevents clearance of misfolded proteins from vacuole membranes and promotes organelle permeability and cell death. We propose that HOPS coordinates Rab, kinase, and SNARE cycles to modulate ILF size during vacuole fusion, regulating lipid and protein turnover important for quality control and membrane integrity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Cdc42 GTPase plays a central role in polarity development in many species. In budding yeast, Cdc42 is essential for polarized growth at the proper site and also for spontaneous cell polarization in the absence of spatial cues. Cdc42 polarization is critical for multiple events in the G1 phase prior to bud emergence, including bud-site assembly, polarization of the actin cytoskeleton, and septin filament assembly to form a ring at the new bud site. Yet the mechanism by which Cdc42 polarizes is not fully understood. Here we report that biphasic Cdc42 polarization in the G1 phase is coupled to stepwise assembly of the septin ring for bud emergence. We show that the Rsr1 GTPase shares a partially redundant role with Gic1 and Gic2, two related Cdc42 effectors, in the first phase of Cdc42 polarization in haploid cells. We propose that the first phase of Cdc42 polarization is mediated by positive feedback loops that function in parallel-one involving Rsr1 via local activation of Cdc42 in response to spatial cues and another involving Gic1 or Gic2 via reduction of diffusion of active Cdc42.

Autophagy mediates the bulk degradation of cytoplasmic material, particularly during starvation. Upon the induction of autophagy, autophagosomes form a sealed membrane around cargo, fuse with a lytic compartment, and release the cargo for degradation. The mechanism of autophagosome-vacuole fusion is poorly understood, although factors that mediate other cellular fusion events have been implicated. In this study, we developed an in vitro reconstitution assay that enables systematic discovery and dissection of the players involved in autophagosome-vacuole fusion. We found that this process requires the Atg14-Vps34 complex to generate PI3P and thus recruit the Ypt7 module to autophagosomes. The HOPS-tethering complex, recruited by Ypt7, is required to prepare SNARE proteins for fusion. Furthermore, we discovered that fusion requires the R-SNARE Ykt6 on the autophagosome, together with the Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. These findings shed new light on the mechanism of autophagosome-vacuole fusion and reveal that the R-SNARE Ykt6 is required for this process., (© 2018 Bas et al.)

The tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7's ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7's functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.

Lysosomes have an important role in cellular protein and organelle quality control, metabolism, and signaling. On the surface of lysosomes, the PIKfyve/Fab1 complex generates phosphatidylinositol 3,5-bisphosphate, PI-3,5-P 2 , which is critical for lysosomal membrane homeostasis during acute osmotic stress and for lysosomal signaling. Here, we identify the inverted BAR protein Ivy1 as an inhibitor of the Fab1 complex with a direct influence on PI-3,5-P 2 levels and vacuole homeostasis. Ivy1 requires Ypt7 binding for its function, binds PI-3,5-P 2 , and interacts with the Fab1 kinase. Colocalization of Ivy1 and Fab1 is lost during osmotic stress. In agreement with Ivy1's role as a Fab1 regulator, its overexpression blocks Fab1 activity during osmotic shock and vacuole fragmentation. Conversely, loss of Ivy1, or lateral relocalization of Ivy1 on vacuoles away from Fab1, results in vacuole fragmentation and poor growth. Our data suggest that Ivy1 modulates Fab1-mediated PI-3,5-P 2 synthesis during membrane stress and may allow adjustment of the vacuole membrane environment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

During autophagy, a newly formed double membrane surrounds its cargo to generate the so-called autophagosome, which then fuses with a lysosome after closure. Previous work implicated that endosomal Rab7/Ypt7 associates to autophagosomes prior to their fusion with lysosomes. Here, we unravel how the Mon1-Ccz1 guanosine exchange factor (GEF) acting upstream of Ypt7 is specifically recruited to the pre-autophagosomal structure under starvation conditions. We find that Mon1-Ccz1 directly binds to Atg8, the yeast homolog of the members of the mammalian LC3 protein family. This requires at least one LIR motif in the Ccz1 C-terminus, which is essential for autophagy but not for endosomal transport. In agreement, only wild-type, but not LIR-mutated Mon1-Ccz1 promotes Atg8-dependent activation of Ypt7. Our data reveal how GEF targeting can specify the fate of a newly formed organelle and provide new insights into the regulation of autophagosome-lysosome fusion., Competing Interests: JG, LL, DK, FR, CU No competing interests declared, (© 2018, Gao et al.)

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Loss-of-function mutations in human endosomal Na + (K + )/H + exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson syndrome, autism, and attention deficit and hyperactivity disorder. These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not the vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function., (© 2018 Karim and Brett. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The identity of organelles in the endomembrane system of any eukaryotic cell critically depends on the correctly localized Rab GTPase, which binds effectors and thus promotes membrane remodeling or fusion. However, it is still unresolved which factors are required and therefore define the localization of the correct fusion machinery. Using SNARE-decorated proteoliposomes that cannot fuse on their own, we now demonstrate that full fusion activity can be achieved by just four soluble factors: a soluble SNARE (Vam7), a guanine nucleotide exchange factor (GEF, Mon1-Ccz1), a Rab-GDP dissociation inhibitor (GDI) complex (prenylated Ypt7-GDI), and a Rab effector complex (HOPS). Our findings reveal that the GEF Mon1-Ccz1 is necessary and sufficient for stabilizing prenylated Ypt7 on membranes. HOPS binding to Ypt7-GTP then drives SNARE-mediated fusion, which is fully GTP-dependent. We conclude that an entire fusion cascade can be controlled by a GEF., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rab GTPases serve as molecular switches to regulate eukaryotic membrane trafficking pathways. The transport protein particle (TRAPP) complexes activate Rab GTPases by catalyzing GDP/GTP nucleotide exchange. In mammalian cells, there are two distinct TRAPP complexes, yet in budding yeast, four distinct TRAPP complexes have been reported. The apparent differences between the compositions of yeast and mammalian TRAPP complexes have prevented a clear understanding of the specific functions of TRAPP complexes in all cell types. In this study, we demonstrate that akin to mammalian cells, wild-type yeast possess only two TRAPP complexes, TRAPPII and TRAPPIII. We find that TRAPPIII plays a major role in regulating Rab activation and trafficking at the Golgi in addition to its established role in autophagy. These disparate pathways share a common regulatory GTPase Ypt1 (Rab1) that is activated by TRAPPIII. Our findings lead to a simple yet comprehensive model for TRAPPIII function in both normal and starved eukaryotic cells., (© 2018 Thomas et al.)

Ypt1 and Sec4 are essential Rab GTPases that control the early and late stages of the yeast secretory pathway, respectively. A chimera consisting of Ypt1 with the switch I domain of Sec4, Ypt1-SW1 Sec4 , is efficiently activated in vitro by the Sec4 exchange factor, Sec2. This should lead to its ectopic activation in vivo and thereby disrupt membrane traffic. Nonetheless early studies found that yeast expressing Ypt1-SW1 Sec4 as the sole copy of YPT1 exhibit no growth defect. To resolve this conundrum, we have analyzed yeast expressing various levels of Ypt1-SW1 Sec4 We show that even normal expression of Ypt1-SW1 Sec4 leads to kinetic transport defects at a late stage of the pathway, with secretory vesicles accumulating near exocytic sites. Higher levels are toxic. Toxicity is suppressed by truncation of Uso1, a vesicle tether required for endoplasmic reticulum-Golgi traffic. The globular head of Uso1 binds to Ypt1 and its coiled-coil tail binds to the Golgi-associated SNARE, Sed5. We propose that when Uso1 is inappropriately recruited to secretory vesicles by Ypt1-SW1 Sec4 , the extended coiled-coil tail blocks docking to the plasma membrane. This putative inhibitory function could serve to increase the fidelity of vesicle docking., Competing Interests: The authors declare no conflict of interest.

The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography-mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P 2 , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1 Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1 ; however, deleting both genes did not rescue the pah1 Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Endosomes are the major protein-sorting hubs of the endocytic pathway. They sort proteins destined for degradation into internal vesicles while in parallel recycling receptors via tubular carriers back to the Golgi. Tubule formation depends on the Rab7/Ypt7-interacting retromer complex, consisting of the sorting nexin dimer (SNX-BAR) and the trimeric cargo selection complex (CSC). Fusion of mature endosomes with the lysosome-like vacuole also requires Rab7/Ypt7. Here we solve a major problem in understanding this dual function of endosomal Rab7/Ypt7, using a fully reconstituted system, including purified, full-length yeast SNX-BAR and CSC, whose overall structure we present. We reveal that the membrane-active SNX-BAR complex displaces Ypt7 from cargo-bound CSC during formation of recycling tubules. This explains how a single Rab can coordinate recycling and fusion on endosomes., (© 2017 Purushothaman, Arlt, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Membrane fusion at endomembranes requires cross-talk between Rab GTPases and tethers to drive SNARE-mediated lipid bilayer mixing. Several tethers have multiple Rab-binding sites with largely untested function. Here we dissected the lysosomal HOPS complex as a tethering complex with just two binding sites for the Rab7-like Ypt7 protein to determine their relevance for fusion. Using tethering and fusion assays combined with HOPS mutants, we show that HOPS-dependent fusion requires both Rab-binding sites, with Vps39 being the stronger Ypt7 interactor than Vps41. The intrinsic amphipathic lipid packaging sensor (ALPS) motif within HOPS Vps41, a target of the vacuolar kinase Yck3, is dispensable for tethering and fusion but can affect tethering if phosphorylated. In combination, our data demonstrate that a multivalent tethering complex uses its two Rab bindings to determine the place of SNARE assembly and thus fusion at endomembranes., (© 2017 Lürick et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Rab guanosine triphosphatases (GTPases) control cellular trafficking pathways by regulating vesicle formation, transport, and tethering. Rab11 and its paralogs regulate multiple secretory and endocytic recycling pathways, yet the guanine nucleotide exchange factor (GEF) that activates Rab11 in most eukaryotic cells is unresolved. The large multisubunit transport protein particle (TRAPP) II complex has been proposed to act as a GEF for Rab11 based on genetic evidence, but conflicting biochemical experiments have created uncertainty regarding Rab11 activation. Using physiological Rab-GEF reconstitution reactions, we now provide definitive evidence that TRAPPII is a bona fide GEF for the yeast Rab11 homologues Ypt31/32. We also uncover a direct role for Arf1, a distinct GTPase, in recruiting TRAPPII to anionic membranes. Given the known role of Ypt31/32 in stimulating activation of Arf1, a bidirectional cross talk mechanism appears to drive biogenesis of secretory and endocytic recycling vesicles. By coordinating simultaneous activation of two essential GTPase pathways, this mechanism ensures recruitment of the complete set of effectors needed for vesicle formation, transport, and tethering., (© 2016 Thomas and Fromme.)

Ypt/Rab GTPases, key regulators of intracellular trafficking pathways, are activated by guanine-nucleotide exchange factors (GEFs). Here, we identify a novel GEF complex, TRAPP IV, which regulates Ypt1-mediated autophagy. In the yeast Saccharomyces cerevisiae, Ypt1 GTPase is required for the initiation of secretion and autophagy, suggesting that it regulates these two distinct pathways. However, whether these pathways are coordinated by Ypt1 and by what mechanism is still unknown. TRAPP is a conserved modular complex that acts as a Ypt/Rab GEF. Two different TRAPP complexes, TRAPP I and the Trs85-containing TRAPP III, activate Ypt1 in the secretory and autophagic pathways, respectively. Importantly, whereas TRAPP I depletion copies Ypt1 deficiency in secretion, depletion of TRAPP III does not fully copy the autophagy phenotypes of autophagy-specific ypt1 mutations. If GEFs are required for Ypt/Rab function, this discrepancy implies the existence of an additional GEF that activates Ypt1 in autophagy. Trs33, a nonessential TRAPP subunit, was assigned to TRAPP I without functional evidence. We show that in the absence of Trs85, Trs33 is required for Ypt1-mediated autophagy and for the recruitment of core-TRAPP and Ypt1 to the preautophagosomal structure, which marks the onset of autophagy. In addition, Trs33 and Trs85 assemble into distinct TRAPP complexes, and we term the Trs33-containing autophagy-specific complex TRAPP IV. Because TRAPP I is required for Ypt1-mediated secretion, and either TRAPP III or TRAPP IV is required for Ypt1-mediated autophagy, we propose that pathway-specific GEFs activate Ypt1 in secretion and autophagy., (Copyright © 2016 by the Genetics Society of America.)

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., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Intracellular trafficking is an essential and conserved eukaryotic process. Rab GTPases are a family of proteins that regulate and provide specificity for discrete membrane trafficking steps by harnessing a nucleotide-bound cycle. Global proteomic screens have revealed many Rab GTPases as phosphoproteins, but the effects of this modification are not well understood. Using the Saccharomyces cerevisiae Rab GTPase Sec4p as a model, we have found that phosphorylation negatively regulates Sec4p function by disrupting the interaction with the exocyst complex via Sec15p. We demonstrate that phosphorylation of Sec4p is a cell cycle-dependent process associated with cytokinesis. Through a genomic kinase screen, we have also identified the polo-like kinase Cdc5p as a positive regulator of Sec4p phosphorylation. Sec4p spatially and temporally localizes with Cdc5p exclusively when Sec4p phosphorylation levels peak during the cell cycle, indicating Sec4p is a direct Cdc5p substrate. Our data suggest the physiological relevance of Sec4p phosphorylation is to facilitate the coordination of membrane-trafficking events during cytokinesis., (© 2016 Lepore et al.)

In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1., (© 2016 Zick and Wickner. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott-Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization.