Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

The class III phosphatidylinositol 3-kinase (PI3K-III) complex and its phosphorylated lipid product phosphatidylinositol 3-phosphate (PtdIns3P) control the three topologically related membrane-involution processes autophagy, endocytosis, and cytokinesis. The activity of the catalytic unit of PI3K-III complex, the Vacuolar sorting protein 34 (VPS34), depends on the membrane targeting unit Vacuolar sorting protein 15 (VPS15), and the tumor suppressor protein Beclin 1. It is established that the overall activity of VPS34 is positively regulated by Beclin 1, whose positive influence is further controlled through the association with a set of Beclin1 interacting components, which stimulate or inhibit VPS34. The interaction between Beclin 1 and Beclin 1-associated components are controllable and is regulated by phosphorylation in a context-dependent manner. Here, we focus on an emerging concept whereby the activity of the PI3K-III complex is controlled by ubiquitination of Beclin 1 or Beclin 1-associated molecules. In summary, at least three different ubiquitin ligases can affect the positive regulatory function of Beclin 1 towards VPS34, suggesting that ubiquitination is an important and physiologically relevant event in tuning the tumor suppressor function of Beclin 1.     

Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast

A membrane-associated complex composed of the Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vps15p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p PtdIns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vps15 proteins. In contrast, catalytically inactive Vps15p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vps15p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperature-conditional allele of VPS15, in which a shift to the nonpermissive temperature leads to a decrease in cellular PtdIns(3)P levels, indicates that the loss of Vps15p function leads to a defect in activation of Vps34p. In addition, characterization of a temperature-sensitive allele of VPS34 demonstrates that inactivation of Vps34p leads to the immediate missorting of soluble vacuolar proteins (e.g., carboxypeptidase Y) without an apparent defect in the sorting of the vacuolar membrane protein alkaline phosphatase. This rapid block in vacuolar protein sorting appears to be the result of loss of PtdIns 3- kinase activity since cellular PtdIns(3)P levels decrease dramatically in vps34 temperature-sensitive mutant cells that have been incubated at the nonpermissive temperature. Finally, analysis of the defects in cellular PtdIns(3)P levels in various vps15 and vsp34 mutant strains has led to additional insights into the importance of PtdIns(3)P intracellular localization, as well as the roles of Vps15p and Vps34p in vacuolar protein sorting.     

PIK3C3/VPS34 control by acetylation

PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) converts phosphatidylinositol (PtdIns) to phosphatidylinositol-3-phosphate (PtdIns3P), sustaining macroautophagy/autophagy and endosomal transport. So far, facilitating the assembly of the PIK3C3/VPS34-BECN1-PIK3R4/VPS15/p150 core complex at distinct membranes is the only known way to activate PIK3C3/VPS34 in cells. We have recently revealed a novel mechanism that regulates PIK3C3/VPS34 activation; cellular PIK3C3/VPS34 is repressed under nutrient-rich conditions by EP300/p300-mediated acetylation. Following nutrient-deprivation that drops EP300 activity, PIK3C3/VPS34 is liberated by deacetylation. Intriguingly, while deacetylation of the N-terminal K29 residue accounts for core complex formation, deacetylation at the C-terminal K771 site determines the binding of PIK3C3/VPS34 to its substrate PtdIns. In vitro and in cell evidence shows that EP300-dependent acetylation and deacetylation is a switch for turning off/on PIK3C3/VPS34 in which deacetylation of K771 is required for its full activation. This PIK3C3/VPS34 activation mechanism is utilized not only by starvation-induced autophagy but also by autophagy without the involvement of AMPK, MTORC1 or ULK1. These findings suggest an alternative circuit in cells for PIK3C3/VPS34 activation, which is involved in membrane transformations in response to metabolic and nonmetabolic cues.     

Selective autophagy inhibition through disruption of the PIK3C3-containing complex I

ABSTRACT

The PIK3C3/VPS34-containing phosphatidylinositol 3-kinase (PtdIns3K) initiation complex (complex I) is necessary for macroautophagy/autophagy initiation and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-ATG14, while the endosomal trafficking complex (complex II) is necessary for vesicle trafficking and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-UVRAG. This composition difference was exploited to identify novel and specific autophagy inhibitors that disrupted the BECN1-ATG14 protein-protein interaction, without affecting vesicle trafficking. A cellular NanoBRET assay was implemented to identify these inhibitors, and one compound was able to successfully disrupt the BECN1-ATG14 interaction and inhibit autophagy, with limited impact on vesicle trafficking. These results reveal the first protein-protein interaction inhibitor targeting the autophagy initiation machinery and demonstrate the viability of targeting protein-protein interactions for the discovery of autophagy-specific modulators.     

Development of in vitro PIK3C3/VPS34 complex protein assay for autophagy-specific inhibitor screening

Autophagy is an important catabolic program to respond to a variety of cellular stresses by forming a double membrane vesicle, autophagosome. Autophagy plays key roles in various cellular functions. Accordingly, dysregulation of autophagy is closely associated with diseases such as diabetes, neurodegenerative diseases, cardiomyopathy, and cancer. In this sense, autophagy is emerging as an important therapeutic target for disease control. Among the autophagy machineries, PIK3C3/VPS34 complex functions as an autophagy-triggering kinase to recruit the subsequent autophagy protein machineries on the phagophore membrane. Accumulating evidence showing that inhibition of PIK3C3/VPS34 complex successfully inhibits autophagy makes the complex an attractive target for developing autophagy inhibitors. However, one concern about PIK3C3/VPS34 complex is that many different PIK3C3/VPS34 complexes have distinct cellular functions. In this study, we have developed an in vitro PIK3C3/VPS34 complex monitoring assay for autophagy inhibitor screening in a high-throughput assay format instead of targeting the catalytic activity of the PIK3C3/VPS34 complex, which shuts down all PIK3C3/VPS34 complexes. We performed in vitro reconstitution of an essential autophagy-promoting PIK3C3/VPS34 complex, Vps34–Beclin1–ATG14L complex, in a microwell plate (96-well format) and successfully monitored the complex formation in many different conditions. This PIK3C3/VPS34 complex protein assay would provide a reliable tool for the screening of autophagy-specific inhibitors.     

Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1

The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p) . Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.     

The G-Protein Rab5A Activates VPS34 Complex II,a Class III PI3K,by a Dual Regulatory Mechanism

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.     

Vac1p coordinates Rab and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome

The vacuolar protein sorting (VPS) pathway of Saccharomyces cerevisiae mediates transport of vacuolar protein precursors from the late Golgi to the lysosome-like vacuole. Sorting of some vacuolar proteins occurs via a prevacuolar endosomal compartment and mutations in a subset of VPS genes (the class D VPS genes) interfere with the Golgi-to-endosome transport step. Several of the encoded proteins, including Pep12p/Vps6p (an endosomal target (t) SNARE) and Vps45p (a Sec1p homologue), bind each other directly [1]. Another of these proteins, Vac1p/Pep7p/Vps19p, associates with Pep12p and binds phosphatidylinositol 3-phosphate (PI(3)P), the product of the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) [1] [2]. Here, we demonstrate that Vac1p genetically and physically interacts with the activated, GTP-bound form of Vps21p, a Rab GTPase that functions in Golgi-to-endosome transport, and with Vps45p. These results implicate Vac1p as an effector of Vps21p and as a novel Sec1p-family-binding protein. We suggest that Vac1p functions as a multivalent adaptor protein that ensures the high fidelity of vesicle docking and fusion by integrating both phosphoinositide (Vps34p) and GTPase (Vps21p) signals, which are essential for Pep12p- and Vps45p-dependent targeting of Golgi-derived vesicles to the prevacuolar endosome.     

Membrane transport in Caenorhabditis elegans: an essential role for VPS34 at the nuclear membrane

Here we present a detailed genetic analysis of let-512/vps34 that encodes the Caenorhabditis elegans homologue of the yeast phosphatidylinositol 3-kinase Vps34p. LET-512/VPS34 has essential functions and is ubiquitously expressed in all tissues and developmental stages. It accumulates at a perinuclear region, and mutations in let-512/vps34 result in an expansion of the outer nuclear membrane as well as in a mislocalization and subsequent complete lack of expression of LRP-1, a C.elegans LDL receptor normally associated with the apical surface of hypodermal cells. Using a GFP::2xFYVE fusion protein we found that the phosphatidylinositol 3-phosphate (PtdIns 3-P) product of LET-512/VPS34 is associated with a multitude of intracellular membranes and vesicles located at the periphery, including endocytic vesicles. We propose that LET-512/VPS34 is required for membrane transport from the outer nuclear membrane towards the cell periphery. Thus, LET-512/VPS34 may regulate the secretory pathway in a much broader range of compartments than was previously suggested for the yeast orthologue.     

Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.     

The C terminus of the Vps34p phosphoinositide 3-kinase is necessary and sufficient for the interaction with the Vps15p protein kinase.

Vps34p is a phosphatidylinositol 3-kinase that is part of a membrane-associated complex with the Vps15p protein kinase. This kinase complex is required for the delivery of soluble proteins to the lysosomal/vacuolar compartment of eukaryotic cells. This study examined the Vps34p-Vps15p association and identified the domains within each protein that were important for this interaction. Using several different approaches, the interaction domain within Vps34p was mapped to a 28-amino acid element near its C terminus. This Vps34p motif was both necessary and sufficient for the interaction with Vps15p. Two-hybrid mapping experiments indicated that two separate regions of Vps15p were required for the association with Vps34p; they are the N-terminal protein kinase domain and a set of three tandem repeats of about 39 amino acids each. Neither domain alone was sufficient for the interaction. These Vps15p repeat elements are similar in sequence to the HEAT motifs that have been implicated in protein interactions in other proteins, including the Huntingtin protein. Finally, these studies identified a novel motif at the very C terminus of Vps34p that was required for phosphatidylinositol 3-kinase activity. This domain is highly conserved specifically in all Vps34p-like phosphatidylinositol 3-kinases but is not required for the interaction with Vps15p. This study thus represents a first step toward a better understanding of how this Vps15p.Vps34p kinase complex is assembled and regulated in vivo.     

人同型融合和蛋白质分选复合体亚基的原核表达、蛋白纯化及功能验证

同型融合和蛋白质分选复合体(HOPS)由VPS11、VPS16、VPS18、VPS33、VPS39和VPS41这6种蛋白组成,能够通过膜融合机制来调节生物体内的膜泡运输。已有研究表明其可以作为融合因子来促进自噬体与溶酶体膜融合过程。为在体外确定HOPS复合体与自噬性SNARE蛋白STX17是否具有直接相互作用,首先利用PCR技术从已有质粒中扩增得到6种基因的编码序列,将其连接至pGEX 4T-1-GST或pET-His-NusA原核表达载体上,经菌落PCR初步鉴定和DNA测序无误后成功构建6种原核表达重组质粒并转化至大肠杆菌BL21(DE3);利用谷胱甘肽琼脂糖树脂与镍柱对重组蛋白进行纯化,烟草蚀纹病毒(TEV)蛋白酶酶切掉GST或His-NusA标签,得到分子量约为105 kDa的HA-VPS11蛋白、97 kDa的Flag-VPS16蛋白、108 kDa的HA-VPS18蛋白、70 kDa的Flag-VPS33蛋白、97 k Da的HA-VPS39蛋白和98 kDa的Flag-VPS41蛋白;通过体外GST pull-down技术对6种蛋白的功能进行验证,证实自噬性SNARE蛋白S...     

A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole.

The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) are required for the sorting of soluble hydrolases to the yeast vacuole. Over-production of Vps34p suppresses the growth and vacuolar protein sorting defects associated with vps15 kinase domain mutants, suggesting that Vps15p and Vps34p functionally interact. Subcellular fractionation and sucrose density gradients indicate that Vps15p is responsible for the association of Vps34p with an intracellular membrane fraction. Chemical cross-linking and native immunoprecipitation experiments demonstrate that Vps15p and Vps34p interact as components of a hetero-oligomeric protein complex. In addition, we show that an intact Vps15 protein kinase domain is required for activation of the Vps34 PI 3-kinase, suggesting that the Vps34 lipid kinase is regulated by a Vps15p-mediated protein phosphorylation event. We propose that Vps15p and Vps34p function together as components of a membrane-associated signal transduction complex that regulates intracellular protein trafficking decisions through protein and lipid phosphorylation events.     

Phosphoproteomic identification of ULK substrates reveals VPS15‐dependent ULK/VPS34 interplay in the regulation of autophagy

Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK‐dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK‐dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation‐independent accumulation of ULK substrates and kinase activity‐regulated recruitment of autophagy proteins to ubiquitin‐positive structures.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.     

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.     

Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on endosomes

Myotubularins constitute a ubiquitous family of phosphatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular myopathy 1 (MTM1)] PI 3-phosphatase is shown associated with early and late endosomes. Loss of endosomal phosphatidylinositol 3-phosphate [PI(3)P] upon overexpression of wild-type MTM1, but not a phosphatase-dead MTM1C375S mutant, resulted in altered early and late endosomal PI(3)P levels and rapid depletion of early endosome antigen-1. Membrane-bound MTM1 was directly complexed to the hVPS15/hVPS34 [vacuolar protein sorting (VPS)] PI 3-kinase complex with binding mediated by the WD40 domain of the hVPS15 (p150) adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P(2) binding. The WD40 domain of hVPS15 also constitutes the binding site for Rab7 and, as shown previously, contributes to Rab5 binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex forms mutually exclusive complexes with the Rab GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab GTPases together with MTM1 likely serve as molecular switches for controlling the sequential synthesis and degradation of endosomal PI(3)P. Normal levels of endosomal PI(3)P and PI(3,5)P(2) are crucial for both endosomal morphology and function, suggesting that disruption of endosomal sorting and trafficking in skeletal muscle when MTM1 is mutated may be a key factor in precipitating X-linked MTM.     

The Saccharomyces cerevisiae protein Ccz1p interacts with components of the endosomal fusion machinery

The yeast protein Ccz1p is necessary for vacuolar protein trafficking and biogenesis. In a complex with Mon1p, it mediates fusion of transport intermediates with the vacuole membrane by activating the small GTPase Ypt7p. Additionally, genetic data suggest a role of Ccz1p in earlier transport steps, in the Golgi. In a search for further proteins interacting with Ccz1p, we identified the endosomal soluble N -ethylmaleimide-sensitive factor attachment protein receptor Pep12p as an interaction partner of Ccz1p. Combining the ccz1 Δ mutation with deletions of PEP12 or other genes encoding components of the endosomal fusion machinery, VPS21, VPS9 or VPS45 , results in synthetic growth phenotypes. The genes MON1 and YPT7 also interact genetically with PEP12 . These results suggest that the Ccz1p–Mon1p–Ypt7p complex is involved in fusion of transport vesicles to multiple target membranes in yeast cells.     

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.     

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein-protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.