Coordination of Golgi functions by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI4Ks) regulate vesicle-mediated export from the Golgi apparatus via phosphatidylinositol 4-phosphate (PtdIns4P) binding effector proteins that control vesicle budding reactions and regulate membrane dynamics. Evidence has emerged from the characterization of Golgi PI4K effectors that vesicle budding and lipid dynamics are tightly coupled via a regulatory network that ensures that the appropriate membrane composition is established before a transport vesicle buds from the Golgi. An important hub of this network is protein kinase D, which regulates the activity of PI4K and several PtdIns4P effectors that control sphingolipid and sterol content of Golgi membranes. Other newly identified PtdIns4P effectors include Vps74/GOLPH3, a phospholipid flippase called Drs2 and Sec2, a Rab guanine nucleotide exchange factor (GEF). These effectors orchestrate membrane transformation events facilitating vesicle formation and targeting. In this review, we discuss how PtdIns4P signaling is integrated with membrane biosynthetic and vesicle budding machineries to potentially coordinate these crucial functions of the Golgi apparatus.     

Local control of phosphatidylinositol 4-phosphate signaling in the Golgi apparatus by Vps74 and Sac1 phosphoinositide phosphatase

In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (K(D) = 3.8 μM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents.     

A Conserved N‐terminal Arginine‐Motif in GOLPH3‐Family Proteins Mediates Binding to Coatomer

Vps74p, a member of the GOLPH3 protein family, binds directly to coatomer and the cytoplasmic tails of a subset of Golgi‐resident glycosyltransferases to mediate their Golgi retention. We identify a cluster of arginine residues at the N‐terminal end of GOLPH3 proteins that are necessary and sufficient to mediate coatomer binding. While loss of coatomer binding renders Vps74p non‐functional for glycosyltransferase retention, the Golgi membrane‐binding capabilities of the mutant protein are not significantly reduced. We establish that the oligomerization status and phosphatidylinositol‐4‐phosphate‐binding properties of Vps74p largely account for the membrane‐binding capacity of the protein and identify an Arf1p–Vps74p interaction as a potential contributing factor in Vps74p Golgi membrane association .     

Golgi Phosphoprotein 3 Triggers Signal-mediated Incorporation of Glycosyltransferases into Coatomer-coated (COPI) Vesicles

Newly synthesized membrane and secreted proteins undergo a series of posttranslational modifications in the Golgi apparatus, including attachment of carbohydrate moieties. The final structure of so-formed glycans is determined by the order of execution of the different glycosylation steps, which seems intimately related to the spatial distribution of glycosyltransferases and glycosyl hydrolases within the Golgi apparatus. How cells achieve an accurate localization of these enzymes is not completely understood but might involve dynamic processes such as coatomer-coated (COPI) vesicle-mediated trafficking. In yeast, this transport is likely to be regulated by vacuolar protein sorting 74 (Vps74p), a peripheral Golgi protein able to interact with COPI coat as well as with a binding motif present in the cytosolic tails of some mannosyltransferases. Recently, Golgi phosphoprotein 3 (GOLPH3), the mammalian homolog of Vps74, has been shown to control the Golgi localization of core 2 N-acetylglucosamine-transferase 1. Here, we highlight a role of GOLPH3 in the spatial localization of α-2,6-sialyltransferase 1. We show, for the first time, that GOLPH3 supports incorporation of both core 2 N-acetylglucosamine-transferase 1 and α-2,6-sialyltransferase 1 into COPI vesicles. Depletion of GOLPH3 altered the subcellular localization of these enzymes. In contrast, galactosyltransferase, an enzyme that does not interact with GOLPH3, was neither incorporated into COPI vesicles nor was dependent on GOLPH3 for proper localization.     

Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components

BACKGROUND: Phosphoinositides are required for the recruitment of many proteins to both the plasma membrane and the endosome; however, their role in protein targeting to other organelles is less clear. The pleckstrin homology (PH) domains of oxysterol binding protein (OSBP) and its relatives have been shown to bind to the Golgi apparatus in yeast and mammalian cells. Previous in vitro binding studies identified phosphatidylinositol (PtdIns) (4)P and PtdIns(4,5)P(2) as candidate ligands, but it is not known which is recognized in vivo and whether phosphoinositide specificity can account for Golgi-specific targeting. RESULTS: We have examined the distribution of GFP fusions to the PH domain of OSBP and to related PH domains in yeast strains carrying mutations in individual phosphoinositide kinases. We find that Golgi targeting requires the activity of the PtdIns 4-kinase Pik1p but not phosphorylation of PtdIns at the 3 or 5 positions and that a PH domain specific for PtdIns(4,5)P(2) is targeted exclusively to the plasma membrane. However, a mutant version of the OSBP PH domain that does not bind phosphoinositides in vitro still shows some targeting in vivo. This targeting is independent of Pik1p but dependent on the Golgi GTPase Arf1p. CONCLUSIONS: Phosphorylation of PtdIns at the 4 position but not conversion to PtdIns(4,5)P(2) contributes to recruitment of PH domains to the Golgi apparatus. However, potential phosphoinositide ligands for these PH domains are not restricted to the Golgi, and the OSBP PH domain also recognizes a second determinant that is ARF dependent, indicating that organelle specificity reflects a combinatorial interaction.     

Sac1–Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.     

Fab1p Is Essential for PtdIns(3)P 5-Kinase Activity and the Maintenance of Vacuolar Size and Membrane Homeostasis

The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH₂-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (Yamamoto et al., 1995). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P₂. Consistent with this, we find that unlike wild-type cells, fab1Δ, fab1^tsf, and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P₂, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P₂ synthesis, on the other hand, is only moderately affected even in fab1Δ mutants. The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P₂ synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P₂. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P₂ by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P₂ has a distinct function. The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size. Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P₂, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P₂.     

Synthetic genetic array analysis of the PtdIns 4-kinase Pik1p identifies components in a Golgi-specific Ypt31/rab-GTPase signaling pathway

Phosphorylated derivatives of phosphatidylinositol are essential regulators of both endocytic and exocytic trafficking in eukaryotic cells. In Saccharomyces cerevisiae, the phosphatidylinositol 4-kinase, Pik1p generates a distinct pool of PtdIns(4)P that is required for normal Golgi structure and secretory function. Here, we utilize a synthetic genetic array analysis of a conditional pik1 mutant to identify candidate components of the Pik1p/PtdIns(4)P signaling pathway at the Golgi. Our data suggest a mechanistic involvement for Pik1p with a specific subset of Golgi-associated proteins, including the Ypt31p rab-GTPase and the TRAPPII protein complex, to regulate protein trafficking through the secretory pathway. We further demonstrate that TRAPPII specifically functions in a Ypt31p-dependent pathway and identify Gyp2p as the first biologically relevant GTPase activating protein for Ypt31p. We propose that multiple stage-specific signals, which may include Pik1p/PtdIns(4)P, TRAPPII and Gyp2p, impinge upon Ypt31 signaling to regulate Golgi secretory function.     

GOLPH3 keeps the Golgi residents at home

In this issue of JCB, Welch et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202106115) show that GOLPH3 mediates the sorting of numerous Golgi proteins into recycling COPI transport vesicles. This explains how many resident proteins are retained at the Golgi and reveals a key role for GOLPH3 in maintaining Golgi homeostasis.

The Golgi apparatus lies at the heart of the secretory pathway, where its major functions are the posttranslational modification of cargo proteins and lipids, particularly at the level of glycosylation, and the sorting of cargo to its correct onward destination. The Golgi is composed of stacked membrane compartments called cisternae, which contain numerous resident enzymes that act on the cargo as it passes through the organelle, from the entry or cis side to the exit or trans side. Each resident enzyme has its own distribution within the Golgi stack, resulting in the sequential modification of the secretory cargo as it moves through the Golgi.Various mechanisms exist to ensure that Golgi residents are retained within the Golgi despite the huge flux of protein and lipid through this organelle (1). Major players are COPI vesicles, which recycle Golgi residents from later to earlier cisternae, at the same time as the cisternae are thought to slowly migrate across the stack, as on a conveyor belt, progressively changing composition in a process referred to as cisternal maturation (2). Unlike the Golgi resident enzymes, which enter recycling vesicles, cargo is thought to remain within the maturing cisternae as it moves through the Golgi. Certain Golgi enzymes can bind directly to the COPI coat, explaining their inclusion in COPI vesicles (3), but for other enzymes and resident proteins, their retention mechanism is less obvious.Previous studies on the peripheral Golgi membrane protein GOLPH3 and its paralogue GOLPH3L (herein I will refer to both proteins as GOLPH3) indicated it can bind to certain Golgi enzymes and to the COPI coat, thereby acting as an adaptor to mediate sorting of these enzymes into COPI vesicles (4, 5). This was first shown for the yeast orthologue Vps74p (6, 7) and has also been demonstrated for the Drosophila version of the protein (8), consistent with a conserved function in Golgi enzyme retention. However, the extent to which GOLPH3 might participate in retention of different Golgi enzymes and other resident proteins, and its importance relative to other methods of protein retention in the Golgi, has remained unclear. Indeed, a recent study suggested that GOLPH3 selectively mediates the retention of enzymes involved in glycosphingolipid synthesis, consistent with a fairly selective role in retaining only a subset of resident Golgi enzymes (9). It should also be noted that GOLPH3 has been implicated in other functions, namely budding of exocytic vesicles from the Golgi, the DNA damage response, and mechanistic target of rapamycin signaling (10).In their current paper, Welch et al. used a combination of approaches to reassess the role of GOLPH3 at the Golgi (11). Using proteomics, they could identify numerous GOLPH3 binding partners, which included COPI, as expected, and a large number of other Golgi residents, including numerous Golgi enzymes and other membrane proteins. The ability of GOLPH3 to retain enzymes at the Golgi was confirmed using microscopy and an innovative flow cytometry–based assay to quantify surface versus Golgi abundance. The large number of possible interactors suggested that GOLPH3 could mediate the Golgi retention of many proteins. To further assess this possibility, the authors took advantage of previous observations showing that Golgi enzymes may be misrouted to the lysosome and degraded upon their failure to be retained in the Golgi (6, 7, 9). Using mass spectrometry, they could show that numerous Golgi resident proteins were depleted in GOLPH3 knockout cells, many of which were also found in the GOLPH3 interactome. This included many enzymes involved in glycosylation, consistent with GOLPH3 playing an important role in maintaining Golgi-dependent glycosylation of proteins and lipids. This was supported by lectin analysis, which showed marked changes in a broad range of glycans in the GOLPH3 knockout cells.The large number of GOLPH3 clients raises the question as to how it can recognize so many proteins. Previous work has shown binding to the cytoplasmic tails of Golgi enzymes and an interaction motif has been described for Vps74p and more recently for GOLPH3 (6, 9). However, bioinformatics analysis of the many GOLPH3 clients combined with mutational analysis, as performed in the current study, revealed the lack of a consensus sequence for GOLPH3 binding, with the common feature being a strong net positive charge combined with short cytoplasmic tail length. This would result in a high positive charge proximal to the membrane, which likely allows interaction with an acidic patch on the surface of GOLPH3. This mode of binding could mediate selective retention of many Golgi residents, while allowing for the forward trafficking of cargo proteins that have longer, less charged, or folded cytoplasmic domains.GOLPH3 is an oncogene associated with many types of cancer (12). Several mechanisms have been proposed to account for the oncogenic properties of GOLPH3, but most compelling is that changes in glycosylation are responsible. It was recently shown that GOLPH3-dependent changes in glycosphingolipids affects cell growth by altering mitogenic signaling (9). Changes in glycosylation of surface receptors has also been reported, which can affect surface abundance and hence signaling (13). The new results from Welch et al. suggest that glycosylation of many proteins and lipids may be relevant in cancer and that potentially a broad range of downstream targets contribute to oncogenesis. Such targets could influence processes beyond signaling, including cell adhesion and migration, that are known to be sensitive to changes in the surface glycome and which have been reported in previous studies on GOLPH3 (12).The study by Welch et al. indicates a major role for GOLPH3 in Golgi protein retention (Fig. 1). Clearly though, other retention mechanisms exist, including direct binding to COPI, and transmembrane domain length is also important, where the short transmembrane domain of resident proteins favors partitioning into recycling COPI vesicles and Golgi cisternal membranes of a similar thickness (1). Additional COPI adaptors are also likely, with TM9SF2 recently identified as a likely candidate, being present in Golgi vesicles and able to bind certain Golgi enzymes (1). It is possible that different resident proteins use different adaptors, or that a combination of retention mechanisms act in conjunction for certain residents, providing robustness to the retention process. However, any redundancy would seem incomplete given the strong phenotype seen upon loss of GOLPH3. GOLPH3 is localized toward the trans side of the Golgi, so it is possible that other adaptors, such as TM9SF2 and possibly others, might act earlier in the Golgi, or that direct coat binding is more important within the early Golgi. Hence different residents may be more likely to use different retention mechanisms depending on their location in the Golgi. Because GOLPH3 acts late in the Golgi and can bind many clients, we may think of it as a gatekeeper to prevent loss of numerous Golgi residents from the organelle.Open in a separate windowFigure 1.GOLPH3 plays a major role in Golgi protein retention. Golgi resident proteins, including many glycosylation enzymes, depicted by lollipops, are sorted into recycling COPI vesicles to maintain retention in the Golgi in the face of onward cisternal maturation and secretory cargo transport. Different enzymes are depicted by different lollipop shapes and colors, with GOLPH3 clients indicated by horizontal ovals. Enzymes retained by other mechanisms are depicted by lollipops with circles (transmembrane domain length), squares or vertical ovals (binding to other COPI adaptors, indicated in turquoise and purple), or hexagons (direct binding to the COPI coat). GOLPH3, which is more abundant toward the trans side of the Golgi, has many clients.With regard to possible future studies, although we have a good idea of how GOLPH3 recognizes its clients, detailed structural analysis will prove informative in elucidating how it can bind so many proteins. Similarly, identification of additional adaptors linking Golgi residents to the COPI coat will be important to generate a more comprehensive view of Golgi protein retention. Finally, in the context of disease, further analysis of the glycoproteins and glycolipids whose levels are altered because of changes in GOLPH3 expression, of which there are likely to be many, should provide significant new insights into the mechanisms underlying GOLPH3-mediated tumorigenesis.     

PtdIns4KIIα generates endosomal PtdIns(4)P and is required for receptor sorting at early endosomes

Phosphatidylinositol 4-kinase IIα (PtdIns4KIIα) localizes to the trans-Golgi network and endosomal compartments and has been implicated in the regulation of endosomal traffic, but the roles of both its enzymatic activity and the site of its action have not been elucidated. This study shows that PtdIns4KIIα is required for production of endosomal phosphatidylinositol 4-phosphate (PtdIns(4)P) on early endosomes and for the sorting of transferrin and epidermal growth factor receptor into recycling and degradative pathways. Depletion of PtdIns4KIIα with small interfering RNA significantly reduced the amount of vesicular PtdIns(4)P on early endosomes but not on Golgi membranes. Cells depleted of PtdIns4KIIα had an impaired ability to sort molecules destined for recycling from early endosomes. We further identify the Eps15 homology domain–containing protein 3 (EHD3) as a possible endosomal effector of PtdIns4KIIα. Tubular endosomes containing EHD3 were shortened and became more vesicular in PtdIns4KIIα-depleted cells. Endosomal PtdIns(4,5)P₂ was also significantly reduced in PtdIns4KIIα-depleted cells. These results show that PtdIns4KIIα regulates receptor sorting at early endosomes through a PtdIns(4)P-dependent pathway and contributes substrate for the synthesis of endosomal PtdIns(4,5)P₂.     

A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments.     

Effects of neuronal PIK3C3/Vps34 deletion on autophagy and beyond

PIK3C3/Vps34 plays important roles in the endocytic and autophagic pathways, both of which are essential for maintaining neuronal integrity. However, it is unclear how inactivating PIK3C3 may affect neuronal endosomal versus autophagic processes in vivo. We generated a conditional null allele of the Pik3c3 gene in mouse, and specifically deleted it in postmitotic sensory neurons. Subsequent analyses reveal several interesting and surprising findings.Key words: PIK3C3/Vps34, ATG7, sensory neurons, neurodegeneration, autophagy, abnormal endosomePIK3C3 (commonly known as Vps34) is the class III phosphatidylinositol 3-kinase (PtdIns3K) that specifically catalyzes the formation of phosphatidylinositol-3-phosphate (PtdIns3P). It is the only PtdIns3K that is conserved from lower eukaryotes to mammals, and represents the most ancient form of PtdIns3Ks. Studies in invertebrate organisms as well as mammalian cell lines show that PIK3C3/Vps34 regulates multiple aspects of both the endocytic and the autophagic pathways. On one hand, PIK3C3 is important for the progression of early endosome to late endosome, and the biogenesis of multivesicular bodies. On the other hand, PIK3C3 is critical for the initiation of autophagosome formation. A chemical inhibitor of PIK3C3, 3-MA, has been commonly used as a specific inhibitor for autophagy. The distinct functions of PIK3C3 are thought to be carried out by at least two different PIK3C3 complexes. In yeast, complex I (Vps34, Vps15, Atg6 and Atg14) is involved in autophagy, whereas complex II (Vps34, Vps15, Atg6 and Vps38) functions in the vacuolar protein sorting process. In mammals, the homologue of complex I (PIK3C3, p150, Beclin 1 and Atg14L) activates autophagy, whereas the homologue of complex II (PIK3C3, p150, Beclin 1 and UVRAG/Vps38) regulates endocytic trafficking.To characterize the in vivo function of PIK3C3 in mammals, we generated a conditional allele of the Pik3c3 gene in mouse and specifically deleted it in postmitotic sensory neurons (Pik3c3-cKO mouse). We focused our analyses on sensory neurons because Pik3c3 is most abundantly expressed in these neurons. Detailed analyses of the sensory ganglia in the knockout mice reveal rapid but differential neurodegenerations of different types of sensory neurons within a few days after birth. Large-diameter myelinated mechanosensory and proprioceptive neurons undergo fast degeneration, whereas mutant small-diameter unmyelinated nociceptive neurons degenerate slower and survive longer.Interestingly, the large-diameter Pik3c3-deleted neurons rapidly accumulate ubiquitin-positive aggregates as well as numerous enlarged vesicles, which are likely abnormal endosomes. The accumulation of enlarged vesicles not only sequesters the cellular membrane source, but also could create trafficking jams that block the transport of prosurvival signals and/or material and organelles, and thus may underlie the rapid demise of large neurons. By contrast, the small-diameter Pik3c3-deleted neurons contain a limited number of vacuoles but gradually build up lysosome- like organelles. The marked increase of lysosomes seems to be more tolerable by neurons, but the mechanism underlying this phenotype is unclear. It could represent a protective and homeostatic response of neurons challenged with stress and insults to their endomembrane system. Alternatively, since sorting of many lysosomal proteins requires PtdIns3P, this phenotype may also result from a build-up of nonfunctional lysosomes as was the case in cathepsin B and L knockout mice. It is also unclear why two types of sensory neurons respond differently to a universal insult. One speculative explanation is that the large-diameter neurons are constantly activated under normal physiological conditions by touch and body movement and thus they contain more active endocytic and membrane trafficking processes; whereas small-diameter pain-sensing neurons are normally not activated and have less endocytic events. These differences might allow the two types of neurons to respond differently to PIK3C3 deletion.We further show that the fast and differential degeneration phenotypes in the Pik3c3-cKO mice are caused primarily by a disruption in the endosomal but not the autophagic pathway. This is validated by comparing the neuronal phenotypes of Pik3c3-cKO mice with those of Atg7-cKO mice, in which the autophagy-specific gene Atg7 is deleted using the same sensory neuron-specific cre driver. Disrupting autophagy leads to a slow degeneration of all types of sensory neurons over a period of several months, and formation of very large intracellular inclusion bodies in all sensory neurons. No increase of lysosomes or accumulation of enlarged vesicles is observed. The completely distinct phenotypes observed in Atg7-cKO versus Pik3c3-cKO mice suggest that inactivation of PIK3C3 primarily disrupts the endosomal pathway rather than inhibiting autophagy (at least in neurons). It calls into attention that care needs to be taken to interpret the results of using PIK3C3 inhibitors such as 3-MA as autophagy-specific inhibitors.The most surprising finding is the existence and activation of a noncanonical, PIK3C3-independent macroautophagy pathway in small-diameter Pik3c3-mutant neurons. Although PIK3C3 is traditionally viewed as indispensable for autophagy initiation, several recent studies suggest a possible PIK3C3-independent autophagy pathway in various cell lines and in Drosophila. We show that this noncanonical autophagy pathway can occur in sensory neurons in vivo using three different assays: crossing Pik3c3-cKO mice to the GFP-LC3 reporter line, western blot analyses of LC3 isoforms, and performing autophagy flux experiments. Interestingly, analyses of Pik3c3/Atg7 double-mutant neurons indicate that this alternative autophagosome initiation pathway still requires ATG7 and hence the conventional conjugation systems. Therefore, this non-canonical autophagy is distinct from the newly reported ATG5/ATG7-independent but PIK3C3-dependent autophagy. We speculate that activation of this PIK3C3-independent autophagy in small-diameter mutant neurons is part of the reason for their longer survival period.The molecular mechanism underlying the PIK3C3-independent autophagosome formation is unknown. It is possible that PtdIns3P can be generated at a low level on the membrane of pre-autophagosomes/phagophores by salvage pathways using other lipid kinases or phosphatases. Alternatively, other mechanisms may direct the formation of the crescent-shaped double membrane structures. For instance, asymmetric insertion into the membrane of proteins with amphipathic helices can induce membrane curvature; BAR domain-containing proteins can also detect and facilitate the formation of curved membrane structures. Thus, these types of proteins might potentially be recruited to nucleate the formation of pre-autophagosomes in the absence of PIK3C3. Finally, the role of this PIK3C3-independent autophagy under normal physiological conditions in vivo needs to be explored.     

An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation

Recently, the Golgi phosphoprotein 3 (GOLPH3) and its yeast homolog Vps74p have been characterized as essential for the Golgi localization of glycosyltransferase in yeast. GOLPH3 has been identified as a new oncogene that is commonly amplified in human cancers to modulate mammalian target of rapamycin signaling. However, the molecular mechanisms of the carcinogenic signaling pathway remain largely unclear. To investigate whether the expression of GOLPH3 was involved in the glycosylation processes in mammalian cells, and whether it affected cell behavior, we performed a loss-of-function study. Cell migration was suppressed in GOLPH3 knockdown (KD) cells, and the suppression was restored by a re-introduction of the GOLPH3 gene. HPLC and LC/MS analysis showed that the sialylation of N-glycans was specifically decreased in KD cells. The specific interaction between sialyltransferases and GOLPH3 was important for the sialylation. Furthermore, overexpression of α2,6-sialyltransferase-I rescued cell migration and cellular signaling, both of which were blocked in GOLPH3 knockdown cells. These results are the first direct demonstration of the role of GOLPH3 in N-glycosylation to regulate cell biological functions.     

Golgi Phosphoprotein 3 Mediates the Golgi Localization and Function of Protein O-Linked Mannose β-1,2-N-Acetlyglucosaminyltransferase 1

GOLPH3 is a highly conserved protein found across the eukaryotic lineage. The yeast homolog, Vps74p, interacts with and maintains the Golgi localization of several mannosyltransferases, which is subsequently critical for N- and O-glycosylation in yeast. Through the use of a T7 phage display, we discovered a novel interaction between GOLPH3 and a mammalian glycosyltransferase, POMGnT1, which is involved in the O-mannosylation of α-dystroglycan. The cytoplasmic tail of POMGnT1 was found to be critical for mediating its interaction with GOLPH3. Loss of this interaction resulted in the inability of POMGnT1 to localize to the Golgi and reduced the functional glycosylation of α-dystroglycan. In addition, we showed that three clinically relevant mutations present in the stem domain of POMGnT1 mislocalized to the endoplasmic reticulum, highlighting the importance of identifying the molecular mechanisms responsible for Golgi localization of glycosyltransferases. Our findings reveal a novel role for GOLPH3 in mediating the Golgi localization of POMGnT1.     

Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus

Phosphatidylinositol 4-kinase, Pik1, is essential for viability. GFP-Pik1 localized to cytoplasmic puncta and the nucleus. The puncta colocalized with Sec7-DsRed, a marker of trans-Golgi cisternae. Kap95 (importin-β) was necessary for nuclear entry, but not Kap60 (importin-α), and exportin Msn5 was required for nuclear exit. Frq1 (frequenin orthologue) also is essential for viability and binds near the NH₂ terminus of Pik1. Frq1-GFP localized to Golgi puncta, and Pik1 lacking its Frq1-binding site (or Pik1 overexpressed in frq1Δ cells) did not decorate the Golgi, but nuclear localization was unperturbed. Pik1(Δ10-192), which lacks its nuclear export sequence, displayed prominent nuclear accumulation and did not rescue inviability of pik1Δ cells. A Pik1-CCAAX chimera was excluded from the nucleus and also did not rescue inviability of pik1Δ cells. However, coexpression of Pik1(Δ10-192) and Pik1-CCAAX in pik1Δ cells restored viability. Catalytically inactive derivatives of these compartment-restricted Pik1 constructs indicated that PtdIns4P must be generated both in the nucleus and at the Golgi for normal cell function.     

白假丝酵母Vps74蛋白的鉴定及其功能

【背景】Vps74/GOLPH3是参与高尔基体蛋白糖基化修饰的关键蛋白,并且是重要的磷酸磷脂酰肌醇效应因子,在胞内参与多种信号通路。【目的】鉴定白假丝酵母Vps74蛋白,并探索其在该病原菌压力应答、蛋白分泌、形态发生及致病过程中的功能。【方法】采用在线序列比对方法,初步鉴定白假丝酵母Vps74蛋白;采用两步PCR介导的同源重组方法,构建白假丝酵母vps74基因缺失菌株vps74Δ/Δ及回补菌株VPS74c;采用反向遗传学方法,探究Vps74在白假丝酵母的压力应答、蛋白分泌、形态发生及致病过程中的功能。【结果】白假丝酵母中存在典型的Vps74/GOLPH3同源蛋白,Vps74参与蛋白糖基化修饰过程,vps74基因缺失导致白假丝酵母蛋白分泌能力、形态发生能力、黏附能力以及侵染宿主能力的显著降低。【结论】Vps74通过影响蛋白分泌、形态发生、黏附、嵌入式生长等过程,在白假丝酵母致病过程中发挥重要作用。     

Phosphatidylinositol 4-Kinases Are Required for Autophagic Membrane Trafficking

Macroautophagy (hereafter autophagy) is a degradative cellular pathway that protects eukaryotic cells from stress, starvation, and microbial infection. This process must be tightly controlled because too little or too much autophagy can be deleterious to cellular physiology. The phosphatidylinositol (PtdIns) 3-kinase Vps34 is a lipid kinase that regulates autophagy, but the role of other PtdIns kinases has not been examined. Here we demonstrate a role for PtdIns 4-kinases and PtdIns4P 5-kinases in selective and nonselective types of autophagy in yeast. The PtdIns 4-kinase Pik1 is involved in Atg9 trafficking through the Golgi and is involved in both nonselective and selective types of autophagy, whereas the PtdIns4P 5-kinase Mss4 is specifically involved in mitophagy but not nonselective autophagy. Our data indicate that phosphoinositide kinases have multiple roles in the regulation of autophagic pathways.     

PtdIns(4,5)P2 and PtdIns3P coordinate to regulate phagosomal sealing for apoptotic cell clearance

Phagocytosis requires phosphoinositides (PIs) as both signaling molecules and localization cues. How PIs coordinate to control phagosomal sealing and the accompanying switch of organelle identity is unclear. In this study, we followed dynamic changes in PIs during apoptotic cell clearance in Caenorhabditis elegans. We found that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂) and phosphatidylinositol-3-phosphate (PtdIns3P), which accumulate transiently on unsealed and fully sealed phagosomes, respectively, are both involved in phagosome closure. We identified PtdIns3P phosphatase MTM-1 as an effector of PtdIns(4,5)P₂ to promote phagosomal sealing. MTM-1 coordinates with the class II PI3 kinase PIKI-1 to control PtdIns3P levels on unsealed phagosomes. The SNX9 family protein LST-4 is required for sealing, and its association with unsealed phagosomes is regulated by PtdIns(4,5)P₂, PIKI-1, and MTM-1. Loss of LST-4 or its retention on phagosomes disrupts sealing and suppresses PtdIns3P accumulation, indicating close coupling of the two events. Our findings support a coincidence detection mechanism by which phagosomal sealing is regulated and coupled with conversion from PtdIns(4,5)P₂ enrichment on unsealed phagosomes to PtdIns3P enrichment on fully sealed phagosomes.     

Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO

Background

Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.

Methods

Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.

Results

Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.

Conclusion

Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.     

Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities.

The Golgi/endosome-associated Vps34 phosphatidylinositol 3-kinase is essential for the sorting of hydrolases from the Golgi to the vacuole/lysosome. Upon inactivation of a temperature-conditional Vps34 kinase, cellular levels of PtdIns(3)P rapidly decrease and it has been proposed that this decrease is due to the continued turnover of PtdIns(3)P by cytoplasmic phosphatases. Here we show that mutations in VAM3 (vacuolar t-SNARE) and YPT7 (rab GTPase), which are required to direct protein and membrane delivery from prevacuolar endosomal compartments to the vacuole, dramatically increase/stabilize PtdIns(3)P levels in vivo by disrupting its turnover. We find that the majority of the total pool of PtdIns(3)P which has been synthesized, but not PtdIns(4)P, requires transport to the vacuole in order to be turned over. Unexpectedly, strains with impaired vacuolar hydrolase activity accumulate 4- to 5-fold higher PtdIns(3)P levels than wild-type cells, suggesting that lumenal vacuolar lipase and/or phosphatase activities degrade PtdIns(3)P. Because vacuolar hydrolases act in the lumen, PtdIns(3)P is likely to be transferred from the cytoplasmic membrane leaflet where it is synthesized, to the lumen of the vacuole. Interestingly, mutants that stabilize PtdIns(3)P accumulate small uniformly-sized vesicles (40-50 nm) within prevacuolar endosomes (multivesicular bodies) or the vacuole lumen. Based on these and other observations, we propose that PtdIns(3)P is degraded by an unexpected mechanism which involves the sorting of PtdIns(3)P into vesicles generated by invagination of the limiting membrane of the endosome or vacuole, ultimately delivering the phosphoinositide into the lumen of the compartment where it can be degraded by the resident hydrolases.