Cloning and characterization of a corepressor and potential component of the nuclear hormone receptor repression complex

Nuclear hormone receptors are potent repressors of transcription in the unliganded state. We describe here the cloning of a nuclear receptor corepressor that we call SUN-CoR (Small Unique Nuclear receptor CoRepressor), which shows no homology to previously described nuclear hormone receptor corepressors, N-CoR, or SMRT. SUN-CoR is a highly basic, 16-kDa nuclear protein that is expressed at high levels in adult tissues and is induced during adipocyte and myogenic differentiation. SUN-CoR potentiates transcriptional repression by thyroid hormone receptor and RevErb in vivo, represses transcription when fused to a heterologous DNA binding domain, and interacts with RevErb as well as with thyroid hormone receptor in vitro. SUN-CoR also interacts with N-CoR and SMRT in vitro and with endogenous N-CoR in cells. We conclude that SUN-CoR is a corepressor and may function as an additional component of the complex involved in transcriptional repression by unliganded and orphan nuclear hormone receptors.

The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis in Dictyostelium discoideum

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

GCS1, an Arf Guanosine Triphosphatase-activating Protein in Saccharomyces cerevisiae, Is Required for Normal Actin Cytoskeletal Organization In Vivo and Stimulates Actin Polymerization In Vitro

Recent cloning of a rat brain phosphatidylinositol 3,4,5-trisphosphate binding protein, centaurin α, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin α is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin α, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Δ) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Δ was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

Glucose-induced Autophagy of Peroxisomes in Pichia pastoris Requires a Unique E1-like Protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Δ had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(ΔATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25–30 kDa protein. This conjugate was not observed in cells expressing Gsa7(ΔATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.

Apg7p/Cvt2p: A Novel Protein-activating Enzyme Essential for Autophagy

In the yeast Saccharomyces cerevisiae, the Apg12p–Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc–tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p–Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p–Apg5p conjugation.

The Thrombospondin Receptor CD47 (IAP) Modulates and Associates with α2β1 Integrin in Vascular Smooth Muscle Cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a β1 integrin, rather than αvβ3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to α2 and β1 integrin subunits and to IAP. A combination of antiα2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antiαvβ3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between α2β1 and IAP was detected by the coimmunoprecipitation of both α2 and β1 integrin subunits with IAP. These data suggest that IAP can associate with α2β1 integrin and modulate its function.

Delayed Activation of the Store-operated Calcium Current Induced by Calreticulin Overexpression in RBL-1 Cells

Calreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation.

Activation of RhoA by Lysophosphatidic Acid and Gα12/13 Subunits in Neuronal Cells: Induction of Neurite Retraction

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein–coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Gα12 and Gα13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity.

A Molecular Network That Produces Spontaneous Oscillations in Excitable Cells of Dictyostelium

A network of interacting proteins has been found that can account for the spontaneous oscillations in adenylyl cyclase activity that are observed in homogenous populations of Dictyostelium cells 4 h after the initiation of development. Previous biochemical assays have shown that when extracellular adenosine 3′,5′-cyclic monophosphate (cAMP) binds to the surface receptor CAR1, adenylyl cyclase and the MAP kinase ERK2 are transiently activated. A rise in the internal concentration of cAMP activates protein kinase A such that it inhibits ERK2 and leads to a loss-of-ligand binding by CAR1. ERK2 phosphorylates the cAMP phosphodiesterase REG A that reduces the internal concentration of cAMP. A secreted phosphodiesterase reduces external cAMP concentrations between pulses. Numerical solutions to a series of nonlinear differential equations describing these activities faithfully account for the observed periodic changes in cAMP. The activity of each of the components is necessary for the network to generate oscillatory behavior; however, the model is robust in that 25-fold changes in the kinetic constants linking the activities have only minor effects on the predicted frequency. Moreover, constant high levels of external cAMP lead to attenuation, whereas a brief pulse of cAMP can advance or delay the phase such that interacting cells become entrained.

αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-αvβ3 antibodies prevent cell adhesion to FGF-2. Also, purified human αvβ3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-αvβ3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with αvβ3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.

Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170’s putative cargo-binding domain. Overexpression studies using p150Glued, the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

The Dynamin-like Protein DLP1 Is Essential for Normal Distribution and Morphology of the Endoplasmic Reticulum and Mitochondria in Mammalian Cells

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.

The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

The modified nucleoside 1-methyladenosine (m1A) is found at position 58 in the TΨC loop of many eukaryotic tRNAs. The absence of m1A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNAiMet). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Δ cells lacked m1A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m1A)methyltransferase [(m1A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m1A in either total tRNA or purified tRNAiMet lacking only this modification. Kinetic analysis of the purified complex revealed KM values for AdoMet or tRNAiMet of 5.0 μM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m1A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein–Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein–Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.

LexA chimeras reveal the function of Drosophila Fos as a context-dependent transcriptional activator

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. Here, we applied it to assay the transactivation potential of proteins in transgenic Drosophila embryos. We found that a chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaved as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) required an unexpected degree of context to function as a transcriptional activator. We provide evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, our data suggest that context-dependence of transcription factors may be more prevalent than previously thought.