Identification of a Saccharomyces cerevisiae Gene that Is Required for G1 Arrest in Response to the Lipid Oxidation Product Linoleic Acid Hydroperoxide*

Reactive oxygen species cause damage to all of the major cellular constituents, including peroxidation of lipids. Previous studies have revealed that oxidative stress, including exposure to oxidation products, affects the progression of cells through the cell division cycle. This study examined the effect of linoleic acid hydroperoxide, a lipid peroxidation product, on the yeast cell cycle. Treatment with this peroxide led to accumulation of unbudded cells in asynchronous populations, together with a budding and replication delay in synchronous ones. This observed modulation of G1 progression could be distinguished from the lethal effects of the treatment and may have been due to a checkpoint mechanism, analogous to that known to be involved in effecting cell cycle arrest in response to DNA damage. By examining several mutants sensitive to linoleic acid hydroperoxide, the YNL099c open reading frame was found to be required for the arrest. This gene (designated OCA1) encodes a putative protein tyrosine phosphatase of previously unknown function. Cells lacking OCA1 did not accumulate in G1 on treatment with linoleic acid hydroperoxide, nor did they show a budding, replication, or Start delay in synchronous cultures. Although not essential for adaptation or immediate cellular survival, OCA1 was required for growth in the presence of linoleic acid hydroperoxide, thus indicating that it may function in linking growth, stress responses, and the cell cycle. Identification of OCA1 establishes cell cycle arrest as an actively regulated response to oxidative stress and will enable further elucidation of oxidative stress-responsive signaling pathways in yeast.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Sterol Biosynthesis Is Required for Heat Resistance but Not Extracellular Survival in Leishmania

Sterol biosynthesis is a crucial pathway in eukaryotes leading to the production of cholesterol in animals and various C24-alkyl sterols (ergostane-based sterols) in fungi, plants, and trypanosomatid protozoa. Sterols are important membrane components and precursors for the synthesis of powerful bioactive molecules, including steroid hormones in mammals. Their functions in pathogenic protozoa are not well characterized, which limits the development of sterol synthesis inhibitors as drugs. Here we investigated the role of sterol C14α-demethylase (C14DM) in Leishmania parasites. C14DM is a cytochrome P450 enzyme and the primary target of azole drugs. In Leishmania, genetic or chemical inactivation of C14DM led to a complete loss of ergostane-based sterols and accumulation of 14-methylated sterols. Despite the drastic change in lipid composition, C14DM-null mutants (c14dm ⁻) were surprisingly viable and replicative in culture. They did exhibit remarkable defects including increased membrane fluidity, failure to maintain detergent resistant membrane fraction, and hypersensitivity to heat stress. These c14dm ⁻ mutants showed severely reduced virulence in mice but were highly resistant to itraconazole and amphotericin B, two drugs targeting sterol synthesis. Our findings suggest that the accumulation of toxic sterol intermediates in c14dm ⁻ causes strong membrane perturbation and significant vulnerability to stress. The new knowledge may help improve the efficacy of current drugs against pathogenic protozoa by exploiting the fitness loss associated with drug resistance.     

ESCRT-0 Is Not Required for Ectopic Notch Activation and Tumor Suppression in Drosophila

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis.     

Plasmodium berghei Calcium Dependent Protein Kinase 1 Is Not Required for Host Cell Invasion

Plasmodium Calcium Dependent Protein Kinase (CDPK1) is required for the development of sexual stages in the mosquito. In addition, it is proposed to play an essential role in the parasite’s invasive stages possibly through the regulation of the actinomyosin motor and micronemal secretion. We demonstrate that Plasmodium berghei CDPK1 is dispensable in the parasite’s erythrocytic and pre-erythrocytic stages. We successfully disrupted P. berghei CDPK1 (PbCDPK1) by homologous recombination. The recovery of erythrocytic stage parasites lacking PbCDPK1 (PbCDPK1-) demonstrated that PbCDPK1 is not essential for erythrocytic invasion or intra-erythrocytic development. To study PbCDPK1’s role in sporozoites and liver stage parasites, we generated a conditional mutant (CDPK1 cKO). Phenotypic characterization of CDPK1 cKO sporozoites demonstrated that CDPK1 is redundant or dispensable for the invasion of mammalian hepatocytes, the egress of parasites from infected hepatocytes and through the subsequent erythrocytic cycle. We conclude that P. berghei CDPK1 plays an essential role only in the mosquito sexual stages.     

Tyrosylprotein Sulfotransferase-2 Expression Is Required for Sulfation of RNase 9 and Mfge8 in Vivo

Protein-tyrosine sulfation is mediated by two Golgi tyrosyl-protein sulfotransferases (TPST-1 and TPST-2) that are widely expressed in vivo. However, the full substrate repertoire of this enzyme system is unknown and thus, our understanding of the biological role(s) of tyrosine sulfation is limited. We reported that whereas Tpst1^-/- male mice have normal fertility, Tpst2^-/- males are infertile despite normal spermatogenesis. However, Tpst2^-/- sperm are severely defective in their motility in viscous media and in their ability to fertilize eggs. These findings suggest that sulfation of unidentified substrate(s) is crucial for normal sperm function. We therefore sought to identify tyrosine-sulfated proteins in the male genital tract using affinity chromatography on PSG2, an anti-sulfotyrosine monoclonal antibody, followed by mass spectrometry. Among the several candidate tyrosine-sulfated proteins identified, RNase 9 and Mfge8 were examined in detail. RNase 9, a catalytically inactive RNase A family member of unknown function, is expressed only in the epididymis after onset of sexual maturity. Mfge8 is expressed on mouse sperm and Mfge8^-/- male mice are subfertile. Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated and both proteins are expressed at comparable levels in wild type, Tpst1^-/-, and Tpst2^-/- epididymides. However, we demonstrate that RNase 9 and Mfge8 are tyrosine-sulfated in wild type and Tpst1^-/-, but not in Tpst2^-/- mice. These findings suggest that lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2^-/- males.Protein-tyrosine sulfation is a post-translational modification described over 50 years ago (1). Tyrosine-sulfated proteins and/or tyrosylprotein sulfotransferase activity have been described in many species in the plant and animal kingdoms (2, 3). In humans, dozens of tyrosine-sulfated proteins have been identified. These include certain adhesion molecules, G-protein-coupled receptors, coagulation factors, serpins, extracellular matrix proteins, hormones, and others. It has been demonstrated that some of these proteins require tyrosine sulfation for optimal function (3).In mice and humans, protein-tyrosine sulfation is mediated by one of two tyrosylprotein sulfotransferases called TPST-1² and TPST-2 (4–6). Mouse TPST-1 and TPST-2 are 370- and 376-residue type II transmembrane proteins, respectively. Each has a short N-terminal cytoplasmic domain followed by a single ≈17-residue transmembrane domain, a membrane proximal ≈40-residue stem region, and a luminal catalytic domain containing four conserved Cys residues and two N-glycosylation sites. The amino acid sequence of human and mouse TPST-1 are ≈96% identical and human and mouse TPST-2 have a similar degree of identity. TPST-1 is ≈65–67% identical to TPST-2 in both mice and humans. TPST-1 and TPST-2 are broadly expressed in human and murine tissues and cell lines and are co-expressed in most, if not all, cell types (3).A variety of biochemical studies have shown that protein-tyrosine sulfation occurs exclusively in the trans-Golgi network (7, 8). This conclusion has been strengthened by more recent immunofluorescence studies showing that a TPST-1/enhanced green fluorescent protein fusion protein co-localizes with golgin-97, a marker for the trans-Golgi network (9). Thus, protein-tyrosine sulfation occurs only on proteins that transit the secretory pathway and occurs well after protein folding and disulfide formation are complete and after N- and O-linked glycosylation are initiated.To gain an understanding of the biological importance of TPSTs, we have generated TPST-deficient mice by targeted disruption of either the Tpst1 or Tpst2 gene. Our studies of Tpst1^-/- mice revealed unexpected but modest effects on body weight and fecundity (10). Tpst1^-/- mice appear healthy but have ≈5% lower average body weight than wild type mice. Fertility of Tpst1^-/- males and females per se was normal. However, Tpst1^-/- females have smaller litters than wild type females due to embryonic lethality between 8.5 and 15.5 days post coitum.In our studies of Tpst2^-/- mice we found that Tpst2^-/- males were infertile, in contrast to Tpst1^-/- males that have normal fertility (11). We found that Tpst2^-/- males were eugonadal and have normal spermatogenesis. Epididymal sperm from Tpst2^-/- males were normal in number, morphology, and motility and appeared to capacitate in vitro and undergo acrosome exocytosis in response to agonist. However, Tpst2^-/- sperm are severely defective in motility in viscous media and in their ability to fertilize zona pellucida (ZP)-intact eggs. In addition, in vitro fertilization experiments revealed that Tpst2^-/- sperm had reduced ability to adhere to the egg plasma membrane, but were able to undergo membrane fusion with the egg.These findings suggest that tyrosine sulfation of one or more substrates is crucial for normal sperm function. However, there are no proteins directly involved in sperm function that are known to be tyrosine-sulfated. The luteinizing hormone receptor and follicle-stimulating hormone receptor are the only proteins important in reproductive biology that are known to be tyrosine-sulfated. Both receptors have been shown to be sulfated at a membrane proximal site in their respective N-terminal extracellular domains that are conserved in many species including the mouse (12). Sulfation of these receptors has been shown to be required for optimal affinity of their cognate ligands in vitro. However, our observations that serum LH, FSH, and testosterone levels are normal in Tpst2^-/- males coupled with the observation that spermatogenesis is normal excludes defective sulfation of these receptors as an explanation for infertility of Tpst2^-/- males (11).In this study, we sought to identify tyrosine-sulfated proteins expressed in the male genital tract that may provide clues as to the mechanism for the infertility of Tpst2^-/- male mice. Among the several candidate tyrosine-sulfated proteins that were identified, RNase 9 and Mfge8 were of particular interest. RNase 9 is a catalytically inactive RNase A family member of unknown function and is expressed only in the epididymis after onset of sexual maturity (13). Mfge8 is expressed on mouse sperm and Mfge8^-/- male mice have been reported to be subfertile (14). Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated. We also showed that both proteins are expressed at comparable levels in wild type, Tpst1^-/-, and Tpst2^-/- epididymides, and that RNase 9 and Mfge8 are sulfated in wild type and Tpst1^-/- mice, but not in Tpst2^-/- mice. Therefore, lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2^-/- male mice.     

Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila

The melanization reaction is a major immune response in Arthropods and involves the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones, which subsequently polymerize into melanin. The Drosophila genome encodes three POs, which are primarily produced as zymogens or prophenoloxidases (PPO). Two of them, PPO1 and PPO2, are produced by crystal cells. Here we have generated flies carrying deletions in PPO1 and PPO2. By analyzing these mutations alone and in combination, we clarify the functions of both PPOs in humoral melanization. Our study shows that PPO1 and PPO2 are responsible for all the PO activity in the hemolymph. While PPO1 is involved in the rapid early delivery of PO activity, PPO2 is accumulated in the crystals of crystal cells and provides a storage form that can be deployed in a later phase. Our study also reveals an important role for PPO1 and PPO2 in the survival to infection with Gram-positive bacteria and fungi, underlining the importance of melanization in insect host defense.     

PRDM12 Is Required for Initiation of the Nociceptive Neuron Lineage during Neurogenesis

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pelo Is Required for High Efficiency Viral Replication

Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection.     

Sgt1 Dimerization Is Required for Yeast Kinetochore Assembly

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1–211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.Spindle microtubules are coupled to the centromeric region of the chromosome by a structural protein complex called the kinetochore (1, 2). The kinetochore is thought to generate a signal that arrests cells during mitosis when it is not properly attached to microtubules, thereby preventing aberrant chromosome transmission to the daughter cells, which can lead to tumorigenesis (3, 4). The kinetochore of the budding yeast Saccharomyces cerevisiae has been characterized thoroughly, genetically and biochemically; thus, its molecular structure is the most well detailed to date. More than 70 different proteins comprise the budding yeast kinetochore, and several of those are conserved in mammals (2).The budding yeast centromere DNA is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEI is bound by Cbf1 (7–9). CDEIII (25 bp) is essential for centromere function (10) and is the site where CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13 (11–18), and Skp1 (17, 18), all of which are essential for viability. Mutations in any of the four CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (19, 20). All of the described kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores dependent on the CBF3 complex (2). Therefore, the CBF3 complex is the fundamental structure of the kinetochore, and the mechanism of CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4 kinetochore-defective mutant dosage suppressor (21). Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required for the formation of the Skp1-Ctf13 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction, the tetratricopeptide repeat (TPR)² (21) and the CS (CHORD protein- and Sgt1-specific) motif. We and others (23–26) have found that both domains are important for the interaction with Hsp90. The Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore complex; this interaction is an initial step in kinetochore assembly (24, 26, 27) that is conserved between yeast and humans (28, 29).In this study, we further characterized the molecular mechanism of this assembly process. We found that Sgt1 forms dimers in vivo, and our results strongly suggest that Sgt1 dimerization is required for kinetochore assembly in budding yeast.     

Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.Influenza A virus is a member of the Orthomyxoviridae and contains a segmented, negative-sense RNA genome that codes for 10 or 11 proteins, depending upon the virus strain (11). The integral membrane protein M2 is the viral ion channel protein that is required during virus entry (29) and for the production of infectious virus particles (4, 10, 12, 13). The sequences responsible for the latter map to the cytoplasmic tail of the protein and overlap with a number of sites for posttranslational modification, which include palmitoylation and phosphorylation (7, 26, 31). Palmitoylation occurs on the cysteine present at amino acid 50 and is not required for ion channel activity of the M2 protein from A/Udorn/72 (H3N2) (7). Palmitoylation of M2 appeared to be dispensable for the production of infectious virus particles using a reassortant virus consisting of seven segments from an H3N8 subtype virus (A/Equine/Miami/63) and the M segment from an H1N1 subtype virus (A/Puerto Rico/8/34) (2). No studies examining the role of M2 palmitoylation in the context of a naturally occurring influenza A virus strain have been published to date.The significance of palmitoylation of the influenza A virus hemagglutinin (HA) protein can vary among virus strains. Palmitoylation of HA from an H7 and an H1 but not an H3 subtype is required for efficient membrane fusion (5, 24, 32), whereas palmitoylation of HA from an H3 but not an H1 subtype is required for virus assembly (5). An analysis of 3,532 sequences of influenza isolates from humans revealed that the M2 residue C50 is conserved in a strain-specific manner. A total of 2,602 of 2,610 H3N2 sequences code for a cysteine at this position; the cysteine, however, is conserved in only 330 of 1,051 H1N1 sequences (data not shown). A serine residue is substituted for cysteine in the majority of the H1N1 viruses that do not have a cytoplasmic palmitoylation site; the newly emerged 2009 H1N1 influenza A viruses, however, do have a cysteine at this position (3). The sequence alignment data are consistent with a strain-specific selective pressure to maintain the palmitoylation site on the M2 protein. Interestingly, other M2 cytoplasmic tail sequences display differential effects on infectious virus production, depending on the strain used (12).To investigate the role of M2 palmitoylation in influenza A virus replication, we substituted a serine for the cysteine residue at position 50 (C50S) of the M2 protein in two influenza A virus strains, A/Udorn/72 (H3N2) (rUdorn) and A/WSN/33 (H1N1) (rWSN). The resultant viruses were tested for their ability to replicate in tissue culture cells, and the mouse-adapted virus was tested for virulence in a mouse model of infection. Neither mutant virus showed any defect in virus replication in tissue culture cells, in differentiated murine primary trachea epithelial cells (mTEC), or in the lungs of infected mice. The viruses lacking a palmitoylation site, however, did have a modest reduction in virulence, suggesting that M2 palmitoylation is dispensable for in vitro replication but contributes to virus virulence in vivo.     

Mov10 and APOBEC3G Localization to Processing Bodies Is Not Required for Virion Incorporation and Antiviral Activity

Mov10 and APOBEC3G (A3G) localize to cytoplasmic granules called processing bodies (P bodies), incorporate into human immunodeficiency virus type 1 (HIV-1) virions, and inhibit viral replication. The functional relevance of Mov10/A3G P-body localization to virion incorporation and antiviral activity has not been fully explored. We found that a helicase V mutant of Mov10 exhibits significantly reduced localization to P bodies but still efficiently inhibits viral infectivity via virion incorporation. Disruption of the P bodies by DDX6 knockdown also confirmed Mov10 antiviral activity without P-body localization. In addition, overexpression of SRP19, which binds to 7SL RNA, depleted A3G from P bodies but did not affect its virion incorporation. Sucrose gradient sedimentation assays revealed that the majority of Mov10, A3G, HIV-1 RNA, and Gag formed high-molecular-mass (HMM) complexes that are converted to low-molecular-mass (LMM) complexes after RNase A treatment. In contrast, the P-body markers DCP2, LSM1, eIF4e, DDX6, and AGO1 were in LMM complexes, whereas AGO2, an effector protein of the RNA-induced silencing complex that localizes to P bodies, was present in both LMM and HMM complexes. Depletion of AGO2 indicated that RNA-induced silencing function is required for Mov10''s ability to reduce Gag expression upon overexpression, but not its virion incorporation or effect on virus infectivity. We conclude that the majority of Mov10 and A3G are in HMM complexes, whereas most of the P-body markers are in LMM complexes, and that virion incorporation and the antiviral activities of Mov10 and A3G do not require their localization to P bodies.     

Nuclear Import Is Required for the Pro-apoptotic Function of the Golgi Protein p115

During apoptosis the Golgi apparatus undergoes irreversible fragmentation. In part, this results from caspase-mediated cleavage of several high molecular weight coiled-coil proteins, termed golgins. These include GM130, golgin 160, and the Golgi vesicle tethering protein p115, whose caspase cleavage generates a C-terminal fragment (CTF) of 205 residues. Here we demonstrate that early during apoptosis, following the rapid cleavage of p115, endogenous CTF translocated to the cell nucleus and its nuclear import was required to enhance the apoptotic response. Expression of a series of deletion constructs identified a putative α-helical region of 26 amino acids, whose expression alone was sufficient to induce apoptosis; deletion of these 26 residues from the CTF diminished its proapoptotic activity. This region contains several potential SUMOylation sites and co-expression of SUMO together with the SUMO ligase, UBC9, resulted in SUMOylation of the p115 CTF. Significantly, when cells were treated with drugs that induce apoptosis, SUMOylation enhanced the efficiency of p115 cleavage and the kinetics of apoptosis. A construct in which a nuclear export signal was fused to the N terminus of p115 CTF accumulated in the cytoplasm and surprisingly, its expression did not induce apoptosis. In contrast, treatment of cells expressing this chimera with the antibiotic leptomycin induced its translocation into the nucleus and resulted in the concomitant induction of apoptosis. These results demonstrate that nuclear import of the p115 CTF is required for it to stimulate the apoptotic response and suggest that its mode of action is confined to the nucleus.In mammalian cells the Golgi apparatus is a highly polarized organelle comprising a series of stacked cisternae, which form a lace-like network in the perinuclear region of the cell. It receives de novo synthesized secretory and membrane proteins, as well as lipids from the endoplasmic reticulum (ER)²; these cargo molecules are then modified, sorted, and transported to lysosomes, endosomes, secretory granules, and the plasma membrane. Although it is well established that the Golgi apparatus undergoes reversible disassembly during mitosis (1, 2), indeed this appears to be a prerequisite for mitosis (3), studies from several laboratories including our own, have also established a link between the Golgi apparatus and apoptosis (programmed cell death). During apoptosis, the Golgi apparatus undergoes extensive and irreversible fragmentation (4), the ER vesiculates (5) and secretion is inhibited (6).Golgi disassembly during apoptosis results, in part, from caspase-mediated cleavage of several golgins (7). Proteolysis of golgin 160 by caspase-2, as well as GRASP65, GM130, p115, syntaxin5, and giantin by caspases-3 and -7 contributes significantly to Golgi fragmentation (6, 8–13). Consistent with this idea, overexpression of caspase-resistant forms of golgin 160, GRASP65, or p115 has been shown to delay the kinetics of Golgi fragmentation during apoptosis (8–10). In addition, immunoreactive caspase-2, an upstream caspase, localizes to the Golgi apparatus (9) and caspase-2-mediated cleavage of golgin 160 also appears to be an early event during apoptosis. Depending on the apoptotic stimulus, expression of a golgin 160 triple mutant resistant to caspase cleavage delays the onset of apoptosis (12). Recently, our laboratory demonstrated that Golgi fragmentation is an early apoptotic event that occurs close to or soon after release of cytochrome c from mitochondria, an early indicator of apoptosis (13). Together these observations demonstrate that specific Golgi proteins may function early during apoptosis, although their role in this process and the detailed molecular mechanism by which Golgi fragmentation occurs is not well understood.A key molecule in mediating Golgi fragmentation during apoptosis is the vesicle tethering protein p115 (10), a 962-residue peripheral membrane protein. p115 is an elongated homodimer consisting of two globular “head” domains, an extended “tail” region reminiscent of the myosin-II structure (14), and 4 sequential coil-coil domains distal to the globular head region, the first of which, CC1, has been implicated in soluble NSF attachment protein receptors (SNARE) binding (15). Earlier in vitro studies on mitotic Golgi reassembly demonstrated that p115 interacts with GM130 and giantin and implicated it in Golgi cisternal stacking (16). Consistent with this idea, microinjection of anti-p115 antibodies caused Golgi fragmentation (17). Based on data demonstrating p115 binding to GM130, giantin, GOS28, and syntaxin-5, Shorter et al. (15) suggested that p115 promotes formation of a GOS28-syntaxin-5 (v-/t-SNARE) complex and hypothesized that it coordinates the sequential tethering and docking of COPI vesicles to Golgi membranes. Interestingly, p115 has also been shown to be a Rab-1 effector that binds Rab-1-GTP directly and cross-linking experiments showed that it interacts with Syntaxin5, sly1, membrin, and rbet1 on microsomal membranes and COPII vesicles suggesting that p115-SNARE interactions may facilitate membrane “docking” (18).More recent in vivo studies showed that inhibition of GM130 or giantin binding to p115 had little effect on Golgi morphology or reassembly following mitosis, suggesting its role in maintaining Golgi structure might be independent of GM130 binding (19, 20). Thus post-mitotic Golgi reassembly could be rescued by p115 lacking the C-terminal GM130 binding motif (residues 935–962) but not by a mutant lacking the SNARE interacting CC1 domain (20). In addition, other studies have implicated GM130 and GRASP65 in Golgi ribbon formation and suggested that this may occur independently of interactions with p115 (21). Most significantly, knockdown of p115 using siRNA demonstrated that it is essential for maintaining Golgi structure, compartmentalization, and cargo traffic to the plasma membrane (20, 22).Earlier work from our laboratory demonstrated that p115 is cleaved in vitro by caspase-8, an initiator caspase, as well as by the executioner caspase-3 (10, 13). In response to apoptosis inducing drugs, p115 is cleaved in vivo at Asp⁷⁵⁷ to generate a 205-residue C-terminal fragment and an N-terminal polypeptide of 757 amino acids. Most significantly, expression of the p115 C-terminal fragment in otherwise healthy cells results in its translocation to the nucleus and the induction of apoptosis suggesting that this polypeptide plays a role in potentiating the apoptotic response. To further dissect p115 function during cell death, we have now determined the minimal domain in its C terminus that mediates apoptosis efficiently and analyzed the requirement of nuclear translocation in triggering the apoptotic response.     

Edin Expression in the Fat Body Is Required in the Defense Against Parasitic Wasps in Drosophila melanogaster

The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae.