ABSTRACT
Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 µM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.
Subject(s)
Cytoskeletal Proteins , Nerve Tissue Proteins , Protein Multimerization , Humans , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/chemistry , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , AnimalsABSTRACT
Activity-regulated cytoskeleton-associated protein (Arc) plays essential roles in diverse forms of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity. In addition, it assembles into virus-like particles that may deliver mRNAs and/or other cargo between neurons and neighboring cells. Considering this broad range of activities, it is not surprising that Arc is subject to regulation by multiple types of post-translational modification, including phosphorylation, palmitoylation, SUMOylation, ubiquitylation, and acetylation. Here we explore the potential regulatory role of Arc phosphorylation by protein kinase C (PKC), which occurs on serines 84 and 90 within an α-helical segment in the N-terminal domain. To mimic the effect of PKC phosphorylation, we mutated the two serines to negatively charged glutamic acid. A consequence of introducing these phosphomimetic mutations is the almost complete inhibition of Arc palmitoylation, which occurs on nearby cysteines and contributes to synaptic weakening. The mutations also inhibit the binding of nucleic acids and destabilize high-order Arc oligomers. Thus, PKC phosphorylation of Arc may limit the full expression of LTD and may suppress the interneuronal transport of mRNAs.
Subject(s)
Lipoylation , Nucleic Acids , Phosphorylation , Protein Processing, Post-Translational , Protein Kinase C/geneticsABSTRACT
Activity-regulated cytoskeletal-associated protein (Arc, also known as activity-regulated gene 3.1 or Arg3.1) is induced in neurons in response to salient experience and neural activity and is necessary for activity-induced forms of synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), cellular substrates of learning and memory. The best-characterized function of Arc is enhancement of the endocytic internalization of AMPA receptors in dendritic spines, a process associated with LTD. Arc has also been implicated in the proteolytic processing of amyloid precursor protein on the surface of endosomes. To mediate these activities, Arc must associate with cellular membranes, but it is unclear whether Arc binds directly to the lipid bilayer or requires protein-protein interactions for membrane recruitment. In this study, we show that Arc associates with pure phospholipid vesicles in vitro and undergoes palmitoylation in neurons, a modification that allows it to insert directly into the hydrophobic core of the bilayer. The palmitoylated cysteines are clustered in a motif, 94CLCRC98, located in the N-terminal half of the protein, which has not yet been structurally characterized. Expression of Arc with three mutated cysteines in that motif cannot support synaptic depression induced by the activity-dependent transcription factor, MEF2 (myocyte enhancer factor 2), in contrast to wild-type Arc. Thus, it appears that palmitoylation regulates at least a subset of Arc functions in synaptic plasticity.
Subject(s)
Cytoskeletal Proteins/metabolism , Lipid Bilayers/metabolism , Lipoylation , Long-Term Synaptic Depression , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Animals , HeLa Cells , Hippocampus/metabolism , Humans , Long-Term Potentiation , Mice , Mice, Inbred C57BL , Neurons/cytology , Palmitates/metabolism , Receptors, AMPA/metabolismABSTRACT
BACKGROUND: Seipin is required for the correct assembly of cytoplasmic lipid droplets. In the absence of the yeast seipin homolog Sei1p (formerly Fld1p), droplets are slow to bud from the endoplasmic reticulum, lack the normal component of proteins on their surface, are highly heterogeneous in size and shape, often bud into the nucleus, and promote local proliferation of the endoplasmic reticulum in which they become tangled. But the mechanism by which seipin catalyzes lipid droplet formation is still uncertain. RESULTS: Seipin prevents a localized accumulation of phosphatidic acid (PA puncta) at ER-droplet junctions. PA puncta were detected with three different probes: Opi1p, Spo20p(51-91) and Pah1p. A system of droplet induction was used to show that PA puncta were not present until droplets were formed; the puncta appeared regardless of whether droplets consisted of triacylglycerol or steryl ester. Deletion strains were used to demonstrate that a single phosphatidic acid-producing enzyme is not responsible for the generation of the puncta, and the puncta remain resistant to overexpression of enzymes that metabolize phosphatidic acid, suggesting that this lipid is trapped in a latent compartment. Suppression of PA puncta requires the first 14 amino acids of Sei1p (Nterm), a domain that is also important for initiation of droplet assembly. Consistent with recent evidence that Ldb16p and Sei1p form a functional unit, the PA puncta phenotype in the ldb16Δ sei1Δ strain was rescued by human seipin. Moreover, PA puncta in the sei1Δ strain expressing Sei1p(ΔNterm) was suppressed by overexpression of Ldb16p, suggesting a functional interaction of Nterm with this protein. Overexpression of both Sei1p and Ldb16p, but not Sei1p alone, is sufficient to cause a large increase in droplet number. However, Ldb16p alone increases triacylglycerol accumulation in the ldb16Δ sei1Δ background. CONCLUSION: We hypothesize that seipin prevents formation of membranes with extreme curvature at endoplasmic reticulum/droplet junctions that would attract phosphatidic acid. While Ldb16p alone can affect triacylglycerol accumulation, proper droplet formation requires the collaboration of Sei1p and Ldb16.
Subject(s)
Endoplasmic Reticulum/metabolism , Lipid Droplets/metabolism , Membrane Proteins/metabolism , Mitochondrial Proteins/metabolism , Phosphatidic Acids/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Triglycerides/metabolism , Biological Transport , Endoplasmic Reticulum/genetics , GTP-Binding Protein gamma Subunits/genetics , GTP-Binding Protein gamma Subunits/metabolism , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mitochondrial Proteins/genetics , Protein Structure, Tertiary , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Purpose: Type 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet ß-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. Results: Here, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 ß-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. Conclusion: Given the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.
Subject(s)
Diabetes Mellitus, Type 1 , Glucagon-Secreting Cells , Islets of Langerhans , Humans , Animals , Glucagon/metabolism , Diabetes Mellitus, Type 1/drug therapy , Diabetes Mellitus, Type 1/metabolism , Insulin/metabolism , Islets of Langerhans/metabolism , Glucagon-Secreting Cells/metabolismABSTRACT
Arc, also known as Arg3.1, is an activity-dependent immediate-early gene product that plays essential roles in memory consolidation. A pool of Arc is located in the postsynaptic cytoplasm, where it promotes AMPA receptor endocytosis and cytoskeletal remodeling. However, Arc is also found in the nucleus, with a major portion being associated with promyelocytic leukemia nuclear bodies (PML-NBs). Nuclear Arc has been implicated in epigenetic control of gene transcription associated with learning and memory. In this study, we use a battery of fluorescence nanoimaging approaches to characterize the behavior of Arc ectopically expressed in heterologous cells. Our results indicate that in the cytoplasm, Arc exists predominantly as monomers and dimers associated with slowly diffusing particles. In contrast, nuclear Arc is almost exclusively monomeric and displays a higher diffusivity than cytoplasmic Arc. We further show that Arc moves freely and rapidly between PML-NBs and the nucleoplasm and that its movement within PML-NBs is relatively unobstructed.
Subject(s)
Cytoskeletal Proteins , Nerve Tissue Proteins , Cell Nucleus/metabolism , Cytoplasm/metabolism , Cytoskeletal Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Receptors, AMPAABSTRACT
Calmodulin kinase-like vesicle-associated (CaMKv), a pseudokinase belonging to the Ca2+/calmodulin-dependent kinase family, is expressed predominantly in brain and neural tissue. It may function in synaptic strengthening during spatial learning by promoting the stabilization and enrichment of dendritic spines. At present, almost nothing is known regarding CaMKv structure and regulation. In this study we confirm prior proteomic analyses demonstrating that CaMKv is palmitoylated on Cys5. Wild-type CaMKv is enriched on the plasma membrane, but this enrichment is lost upon mutation of Cys5 to Ser. We further show that CaMKv interacts with another regulator of synaptic plasticity, Arc/Arg3.1, and that the interaction between these two proteins is weakened by mutation of the palmitoylated cysteine in CamKv.
ABSTRACT
Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.
Subject(s)
Insulin-Secreting Cells , Insulin , Apoptosis , Calcium/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Humans , Insulin/metabolism , Insulin-Secreting Cells/metabolismABSTRACT
The activity-regulated cytoskeletal-associated protein (Arc, also known as Arg3.1) is an immediate early gene product induced by activity/experience and required for multiple modes of synaptic plasticity. Both long-term potentiation (LTP) and long-term depression (LTD) are impaired upon Arc deletion, as well as the ability to form long-term spatial, taste and fear memories. The best-characterized cellular function of Arc is enhancement of the endocytic internalization of AMPA receptors (AMPARs) in dendritic spines. Solution of the crystal structure of a C-terminal segment of Arc revealed a striking similarity to the capsid domain of HIV Gag. It was subsequently shown that Arc assembles into viral capsid-like structures that enclose Arc mRNA, are released into the extracellular space, and are internalized by neighboring cells. Thus, Arc is unique in participating in plasma membrane budding both into and out of the cell. In this report we study the interaction of Arc with membranes using giant unilamellar vesicles (GUVs). Using the fluorescent lipid probe LAURDAN, we find that Arc promotes the formation of smaller vesicles that penetrate into the GUV interior. Our results suggest that Arc induces negative membrane curvature and may therefore facilitate the formation of mRNA-containing extracellular vesicles from the plasma membrane.
ABSTRACT
Mutations in the gene encoding dynamin 2 (DNM2), a GTPase that catalyzes membrane constriction and fission, are associated with two autosomal-dominant motor disorders, Charcot-Marie-Tooth disease (CMT) and centronuclear myopathy (CNM), which affect nerve and muscle, respectively. Many of these mutations affect the pleckstrin homology domain of DNM2, yet there is almost no overlap between the sets of mutations that cause CMT or CNM. A subset of CMT-linked mutations inhibit the interaction of DNM2 with phosphatidylinositol (4,5) bisphosphate, which is essential for DNM2 function in endocytosis. In contrast, CNM-linked mutations inhibit intramolecular interactions that normally suppress dynamin self-assembly and GTPase activation. Hence, CNM-linked DNM2 mutants form abnormally stable polymers and express enhanced assembly-dependent GTPase activation. These distinct effects of CMT and CNM mutations are consistent with current findings that DNM2-dependent CMT and CNM are loss-of-function and gain-of-function diseases, respectively. In this study, we present evidence that at least one CMT-causing DNM2 mutant (ΔDEE; lacking residues 555DEE557) forms polymers that, like the CNM mutants, are resistant to disassembly and display enhanced GTPase activation. We further show that the ΔDEE mutant undergoes 2-3-fold higher levels of tyrosine phosphorylation than wild-type DNM2. These results suggest that molecular mechanisms underlying the absence of pathogenic overlap between DNM2-dependent CMT and CNM should be re-examined.
ABSTRACT
Dynamins induce membrane vesiculation during endocytosis and Golgi budding in a process that requires assembly-dependent GTPase activation. Brain-specific dynamin 1 has a weaker propensity to self-assemble and self-activate than ubiquitously expressed dynamin 2. Here we show that dynamin 3, which has important functions in neuronal synapses, shares the self-assembly and GTPase activation characteristics of dynamin 2. Analysis of dynamin hybrids and of dynamin 1-dynamin 2 and dynamin 1-dynamin 3 heteropolymers reveals that concentration-dependent GTPase activation is suppressed by the C-terminal proline/arginine-rich domain of dynamin 1. Dynamin proline/arginine-rich domains also mediate interactions with SH3 domain-containing proteins and thus regulate both self-association and heteroassociation of dynamins.
Subject(s)
Arginine/chemistry , Dynamins/chemistry , Dynamins/metabolism , Proline/chemistry , Animals , Cell Line , Dynamin I/chemistry , Dynamin I/genetics , Dynamin I/metabolism , Dynamin II/chemistry , Dynamin II/genetics , Dynamin II/metabolism , Dynamin III/chemistry , Dynamin III/genetics , Dynamin III/metabolism , Dynamins/genetics , Humans , Proline-Rich Protein Domains/genetics , Proline-Rich Protein Domains/physiology , Protein Structure, Tertiary , SpodopteraABSTRACT
Adrenergic signaling is a well-known input into pancreatic islet function. Specifically, the insulin-secreting islet ß cell expresses the Gi/o-linked α2-adrenergic receptor, which upon activation suppresses insulin secretion. The use of the adrenergic agonist epinephrine at micromolar doses may have supraphysiological effects. We found that pretreating ß cells with micromolar concentrations of epinephrine differentially inhibited activation of receptor tyrosine kinases. We chose TrkB as an example because of its relative sensitivity to the effects of epinephrine and due to its potential regulatory role in the ß cell. Our characterization of brain-derived neurotrophic factor (BDNF)-TrkB signaling in MIN6 ß cells showed that TrkB is activated by BDNF as expected, leading to canonical TrkB autophosphorylation and subsequent downstream signaling, as well as chronic effects on ß cell growth. Micromolar, but not nanomolar, concentrations of epinephrine blocked BDNF-induced TrkB autophosphorylation and downstream mitogen-activated protein kinase pathway activation, suggesting an inhibitory phenomenon at the receptor level. We determined epinephrine-mediated inhibition of TrkB activation to be Gi/o-dependent using pertussis toxin, arguing against an off-target effect of high-dose epinephrine. Published data suggested that inhibition of potassium channels or phosphoinositide-3-kinase signaling may abrogate the negative effects of epinephrine; however, these did not rescue TrkB signaling in our experiments. Taken together, these results show that (1) TrkB kinase signaling occurs in ß cells and (2) use of epinephrine in studies of insulin secretion requires careful consideration of concentration-dependent effects. BDNF-TrkB signaling in ß cells may underlie pro-survival or growth signaling and warrants further study.
ABSTRACT
Mammalian cells contain two isoforms of the type II PI4K (phosphoinositol 4-kinase), PI4KIIalpha and beta. These 55 kDa proteins have highly diverse N-terminal regions (approximately residues 1-90) but conserved catalytic domains (approximately from residue 91 to the C-termini). Nearly the entire pool of PI4KIIalpha behaves as an integral membrane protein, in spite of a lack of a transmembrane domain. This integral association with membranes is due to palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domain. Although the CCPCC motif is conserved in PI4KIIbeta, only 50% of PI4KIIbeta is membrane-associated, and approximately half of this pool is only peripherally attached to the membranes. Growth factor stimulation or overexpression of a constitutively active Rac mutant induces the translocation of a portion of cytosolic PI4KIIbeta to plasma membrane ruffles and stimulates its activity. Here, we demonstrate that membrane-associated PI4KIIbeta undergoes two modifications, palmitoylation and phosphorylation. The cytosolic pool of PI4KIIbeta is not palmitoylated and has much lower lipid kinase activity than the membrane-associated kinase. Although only membrane-associated PI4KIIbeta is phosphorylated in the unique N-terminal region, this modification apparently does not influence its membrane binding or activity. A series of truncation mutants and alpha/beta chimaeras were generated to identify regions responsible for the isoform-specific behaviour of the kinases. Surprisingly, the C-terminal approx. 160 residues, and not the diverse N-terminal regions, contain the sites that are most important in determining the different solubilities, palmitoylation states and stimulus-dependent redistributions of PI4KIIalpha and beta.
Subject(s)
Cell Membrane/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Binding Sites , COS Cells , Cells, Cultured , Chlorocebus aethiops , HeLa Cells , Humans , Lipoylation , Microscopy, Fluorescence , Minor Histocompatibility Antigens , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Processing, Post-Translational , Transfection , rac GTP-Binding Proteins/metabolismABSTRACT
Pet10p is a yeast lipid droplet protein of unknown function. We show that it binds specifically to and is stabilized by droplets containing triacylglycerol (TG). Droplets isolated from cells with a PET10 deletion strongly aggregate, appear fragile, and fuse in vivo when cells are cultured in oleic acid. Pet10p binds early to nascent droplets, and their rate of appearance is decreased in pet10Δ Moreover, Pet10p functionally interacts with the endoplasmic reticulum droplet assembly factors seipin and Fit2 to maintain proper droplet morphology. The activity of Dga1p, a diacylglycerol acyltransferase, and TG accumulation were both 30-35% lower in the absence of Pet10p. Pet10p contains a PAT domain, a defining property of perilipins, which was not previously known to exist in yeast. We propose that the core functions of Pet10p and other perilipins extend beyond protection from lipases and include the preservation of droplet integrity as well as collaboration with seipin and Fit2 in droplet assembly and maintenance.
Subject(s)
Cation Transport Proteins/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Glycoproteins/metabolism , Lipid Droplets/metabolism , Perilipin-1/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cation Transport Proteins/genetics , GTP-Binding Protein gamma Subunits/genetics , Glycoproteins/genetics , Perilipin-1/genetics , Protein Domains , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Seipin is necessary for both adipogenesis and lipid droplet (LD) organization in nonadipose tissues; however, its molecular function is incompletely understood. Phenotypes in the seipin-null mutant of Saccharomyces cerevisiae include aberrant droplet morphology (endoplasmic reticulum-droplet clusters and size heterogeneity) and sensitivity of droplet size to changes in phospholipid synthesis. It has not been clear, however, whether seipin acts in initiation of droplet synthesis or at a later step. Here we utilize a system of de novo droplet formation to show that the absence of seipin results in a delay in droplet appearance with concomitant accumulation of neutral lipid in membranes. We also demonstrate that seipin is required for vectorial budding of droplets toward the cytoplasm. Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology. Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant. We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.
Subject(s)
GTP-Binding Protein gamma Subunits/physiology , Lipid Droplets/metabolism , Lipid Metabolism , Saccharomyces cerevisiae/metabolism , Cytoplasm/metabolism , Cytoplasm/ultrastructure , Endoplasmic Reticulum/metabolism , GTP-Binding Protein gamma Subunits/genetics , GTP-Binding Protein gamma Subunits/metabolism , Gene Knockout Techniques , Phenotype , Saccharomyces cerevisiae/ultrastructureABSTRACT
Lipins are phosphatidate phosphatases that generate diacylglycerol (DAG). In this study, we report that yeast lipin, Pah1p, controls the formation of cytosolic lipid droplets. Disruption of PAH1 resulted in a 63% decrease in droplet number, although total neutral lipid levels did not change. This was accompanied by an accumulation of neutral lipids in the endoplasmic reticulum (ER). The droplet biogenesis defect was not a result of alterations in neutral lipid ratios. No droplets were visible in the absence of both PAH1 and steryl acyltransferases when grown in glucose medium, even though the strain produces as much triacylglycerol as wild type. The requirement of PAH1 for normal droplet formation can be bypassed by a knockout of DGK1. Nem1p, the activator of Pah1p, localizes to a single punctum per cell on the ER that is usually next to a droplet, suggesting that it is a site of droplet assembly. Overall, this study provides strong evidence that DAG generated by Pah1p is important for droplet biogenesis.
Subject(s)
Cytosol/metabolism , Lipid Metabolism , Lipids/chemistry , Phosphatidate Phosphatase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Diglycerides/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Organic Chemicals/metabolism , Phosphatidate Phosphatase/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) and IIbeta (PI4KIIbeta). Nearly all of PI4KIIalpha, and about half of PI4KIIbeta, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIalpha converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity ( Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708 ). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIalpha to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIalpha in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIalpha and associates more weakly with lipid rafts.
Subject(s)
1-Phosphatidylinositol 4-Kinase/chemistry , Lipoylation , 1-Phosphatidylinositol 4-Kinase/metabolism , Amino Acid Motifs , Animals , COS Cells , Catalysis , Cell Membrane/metabolism , Chlorocebus aethiops , Insecta , Membrane Microdomains/chemistry , Models, Biological , Proline/chemistry , Rats , Recombinant Proteins/chemistry , trans-Golgi Network/metabolismABSTRACT
Dynamin is a large molecular weight GTPase. Amongst other biological processes, it is involved in clathrin-dependent endocytosis. It can self-assemble or assemble on other macromolecular structures that result in an increase in its GTPase activity. Its role in endocytosis has been variously attributed to being a force-generating enzyme or a signalling protein. Here we review evidence for the oligomeric state of dynamin at high and low ionic strength conditions. We also review work on the elementary processes of the dynamin GTPase at high ionic strength and compare these to the ATPase of the force-generating protein myosin and the GTPase of the signalling protein Ras. New data on the interaction of dynamin with a fluorescent derivative of GTPgammaS are also presented. The possible mechanism by which assembly of dynamin leads to an increase in its GTPase activity is discussed.
Subject(s)
Dynamins/chemistry , Dynamins/metabolism , Guanosine Triphosphate/metabolism , Macromolecular Substances , Protein Binding , Protein ConformationABSTRACT
The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5. The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.
Subject(s)
1-Phosphatidylinositol 4-Kinase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , 1-Phosphatidylinositol 4-Kinase/genetics , 1-Phosphatidylinositol 4-Kinase/isolation & purification , Base Sequence , Cloning, Molecular , Kinetics , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Substrate SpecificityABSTRACT
Phosphatidylinositol (PtdIns) 4-kinases catalyze the conversion of PtdIns to PtdIns 4-phosphate, the major precursor of phosphoinositides that regulates a vast array of cellular processes. Based on enzymatic differences, two classes of PtdIns 4-kinase have been distinguished termed Types II and III. Type III kinases, which belong to the phosphatidylinositol (PI) 3/4-kinase family, have been extensively characterized. In contrast, little is known about the Type II enzymes (PI4KIIs), which have been cloned and sequenced very recently. PI4KIIs bear essentially no sequence similarity to other protein or lipid kinases; hence, they represent a novel and distinct branch of the kinase superfamily. Here we define the minimal catalytic domain of a rat PI4KII isoform, PI4KIIalpha, and identify conserved amino acid residues required for catalysis. We further show that the catalytic domain by itself determines targeting of the kinase to membrane rafts. To verify that the PI4KII family extends beyond mammalian sources, we expressed and characterized Drosophila PI4KII and its catalytic domain. Depletion of PI4KII from Drosophila cells resulted in a severe reduction of PtdIns 4-kinase activity, suggesting the in vivo importance of this enzyme.