Isolation of a matrix that binds medial Golgi enzymes

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae.     

Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells.

Osmium impregnation was used to determine the number of Golgi apparatus in both interphase and mitotic HeLa cells. The number was found to increase substantially during mitosis to the point where random partitioning alone would explain the nearly equal numbers found in each daughter cell.     

Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.     

Kin recognition in a social spider

Social spiders accept immigrant spiders into their kin-based groups, suggesting that spiders cannot recognise kin and may lose inclusive fitness benefits. A field and two laboratory experiments on Diaea ergandros, a social crab spider, demonstrated that younger and older instar D. ergandros do discriminate siblings, but potential benefits were variable and not equally distributed. First, proportional survival was greater in large groups regardless of the within-group relatedness, so accepting immigrants increases probability of group survival (although relatedness was more important among smaller groups). Second, juvenile D. ergandros ate unrelated spiders instead of siblings when starved, so immigrants might represent a food reserve in times of food shortage. Third, subadult resident, sibling females cannibalised unrelated, immigrant females and their brothers instead of immigrant males when starved, suggesting that subadult female spiders may maximise outbreeding opportunities. These benefits provide selective pressure for groups to accept immigrants, but as benefits are realised differentially, conflict and cooperation will exist within spider groups similar to that shown in other group-living taxa.     

Kin recognition in an annual plant

Kin recognition is important in animal social systems. However, though plants often compete with kin, there has been as yet no direct evidence that plants recognize kin in competitive interactions. Here we show in the annual plant Cakile edentula, allocation to roots increased when groups of strangers shared a common pot, but not when groups of siblings shared a pot. Our results demonstrate that plants can discriminate kin in competitive interactions and indicate that the root interactions may provide the cue for kin recognition. Because greater root allocation is argued to increase below-ground competitive ability, the results are consistent with kin selection.     

Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells

The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved multi-subunit protein complex that regulates membrane trafficking in eukaryotic cells. In this work we used short interfering RNA strategy to achieve an efficient knockdown (KD) of Cog3p in HeLa cells. For the first time, we have demonstrated that Cog3p depletion is accompanied by reduction in Cog1, 2, and 4 protein levels and by accumulation of COG complex-dependent (CCD) vesicles carrying v-SNAREs GS15 and GS28 and cis-Golgi glycoprotein GPP130. Some of these CCD vesicles appeared to be vesicular coat complex I (COPI) coated. A prolonged block in CCD vesicles tethering is accompanied by extensive fragmentation of the Golgi ribbon. Fragmented Golgi membranes maintained their juxtanuclear localization, cisternal organization and are competent for the anterograde trafficking of vesicular stomatitis virus G protein to the plasma membrane. In a contrast, Cog3p KD resulted in inhibition of retrograde trafficking of the Shiga toxin. Furthermore, the mammalian COG complex physically interacts with GS28 and COPI and specifically binds to isolated CCD vesicles.     

Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway

Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.     

Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block.

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1p(dn) concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.     

Kin and sex recognition in a dioecious grass

Plants have evolved complex mechanisms to recognize and respond to the presence of neighboring plants, and the genetic identity of a neighbor has been shown to make a difference in this response. Studies have found that plants are able to differentiate among self- versus non-self and among sibling (kin) competitors. Here, we present data for the dioecious grass Distichlis spicata on seedling recognition of kin and sex. D. spicata exhibits extreme spatial segregation of the sexes (SSS) in the field, and previous work has shown that intra-sexual competition is less than inter-sexual competition in the field. In this experiment, we conducted experiments in the lab, exposing the seedlings to liquid media in which seedlings had been previously grown, rather than have the seedling physically contact one another. We found that inter-sexual interactions caused a decrease in the total dry weight and an increase in root/shoot ratio of the plants compared with intra-sexual interactions. These findings suggest that D. spicata plants can recognize and respond to plant sex and that inter-sexual competition contributes to SSS, even when additional interactions, such as mycorrhizal fungi are controlled, and physical interactions between plants are removed. In the kin recognition analysis, we found that plants paired with another plant from the same mother had significantly greater lateral root number and length than plants paired with non-kin, suggesting that in this highly clonal grass, kin recognition may be an important mechanism in competitive interactions.     

Heat-shock proteins associated with base excision repair enzymes in HeLa cells

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.     

Biosynthesis and modification of Golgi mannosidase II in HeLa and 3T3 cells

The biosynthesis and post-translational modification of mannosidase II, an enzyme required in the maturation of asparagine-linked oligosaccharides in the Golgi complex, has been investigated. Antibody raised against this enzyme purified from rat liver Golgi membranes was used to immunoprecipitate mannosidase II from rat liver, 3T3 cells, or HeLa cells. Mannosidase II immunoprecipitated from rat liver Golgi membranes, when analyzed by polyacrylamide gel electrophoresis, migrated with an apparent molecular weight of approximately 124,000. In contrast, the enzyme purified from rat liver Golgi membranes was shown to contain both the 124,000-dalton component and a 110,000-dalton polypeptide believed to result from degradation of intact mannosidase II during purification. Mannosidase II from 3T3 and HeLa cells migrated on polyacrylamide gels with apparent molecular weights of approximately 124,000 and 134,000-136,000, respectively. When immunoprecipitated from radiolabeled cultures, mannosidase II from both cell types was similar in the following respects: (a) the initial synthesis product had an apparent molecular weight of approximately 124,000; (b) in cultures treated with tunicamycin the initial synthesis product had an apparent molecular weight of approximately 117,000; (c) endoglycosidase H digestion of the initial synthesis product gave an apparent molecular weight similar to the tunicamycin-induced polypeptide; (d) the mature enzyme was mostly (HeLa) or entirely (3T3) resistant to digestion by endoglycosidase H. Loss of [35S]methionine from intracellular mannosidase II occurred with a half-life of approximately 20 h; there was no appreciable accumulation of labeled immuno-reactive material in the medium. HeLa mannosidase II, but not the 3T3 enzyme, was additionally modified 1-3 h after synthesis, the initial synthesis product being converted to a doublet with an apparent molecular weight of approximately 134,000-136,000. Evidence is presented that this mobility shift may result from O-glycosylation. Mannosidase II from both cell types could be labeled with [32P]phosphate or [35S]sulfate. The latter is apparently attached to oligosaccharide as indicated by inhibition of labeling by tunicamycin; the former was shown with the HeLa enzyme to be present as serine phosphate moieties. In addition, [3H]palmitate could be incorporated into the enzyme in 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells.

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.     

Kin recognition: knowing who's boss in wasp colonies

Paper wasps recognise the dominant individual in their colony and surrender reproduction to this alpha individual. Contrary to expectations her dominance status is not signalled by a chemical indicator of fertility.     

Purification and characterization of primer recognition proteins from HeLa cells

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.     

Kin recognition in the ant Rhytidoponera confusa I. Environmental odour

The relative contribution of genetic and environmental components to the colony-specific recognition odour was assessed for the ant Rhytidoponera confusa, using an aggression assay. Colonies maintained in the laboratory for 6 months were significantly less aggressive to each other than they were to colonies that had been collected from the field within the previous 48 h. Colonies tested 12–24 h after collection from the field showed significantly more aggression against laboratory ants than did the same colonies after they had been kept in the laboratory for 4–5 weeks. Both results indicate that environmental odours can contribute to the colony odour. Nestmates supplied with different diets and nest materials for 4–5 weeks were attacked more than the controls. Though slight, the increase in aggression was significant. This suggests that environmental odours, though significant when odour differences are extreme enough, may nevertheless be less important than genetic odours in nature. This and other studies suggest this pattern may be common in social insects generally.     

Specificities of enzymes of glycosylphosphatidylinositol biosynthesis in Trypanosoma brucei and HeLa cells

A series of synthetic analogues of d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, consisting of 22 variants of the d-GlcN or lipid components, were tested in trypanosomal and human (HeLa) cell-free systems. The assays measured the abilities of the analogues to act as substrates or inhibitors of the enzymes of glycosylphosphatidylinositol biosynthesis downstream of GlcNAc-phosphatidylinositol (GlcNAc-PI) de-N-acetylase. One compound, 4-deoxy-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, proved to be an inhibitor of both the trypanosomal and HeLa pathways, whereas 4-O-methyl-d-GlcN alpha 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol and the 4'-epimer, d-GalN-alpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol, were neither substrates nor inhibitors. The results with other analogues showed that the 6-OH of the alpha-d-GlcN residue is not required for substrate recognition in the trypanosomal and human pathways, whereas the 3-OH group is essential for both. Parasite-specific recognition of the beta-linked analogue d-GlcN beta 1-6-d-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol is striking. This suggests that, like the GlcNAc-PI de-N-acetylase, the trypanosomal glycosylphosphatidylinositol alpha-mannosyltransferases, inositol acyltransferse and ethanolamine phosphate transferase, do not recognize the 2-, 3-, 4-, and 5-OH groups of the d-myo-inositol residue, whereas the human inositol acyltransferase and/or first alpha-mannosyltransferase recognizes one or more of these groups. All of the various lipid analogues tested served as substrates in both the trypanosomal and HeLa cell-free systems, suggesting that a precise lipid structure and stereochemistry are not essential for substrate recognition. However, an analogue containing a single C18:0 alkyl chain in place of sn-1,2-dipalmitoylglycerol proved to be a better substrate in the trypanosomal than in the HeLa cell-free system. These findings should have a bearing on the design of future generations of specific inhibitors of the trypanosomal glycosylphosphatidylinositol biosynthetic pathway.     

Kin recognition and incest avoidance in a group-living insect

Mate choice theories predict that animals evolved strategiesto mate with optimally genetically dissimilar partners, providingfitness benefits. In group-living species, when adults do notdisperse, assessment of relatedness between conspecifics canbe a key factor for choosing mates. Here, we report for thefirst time, kin recognition abilities and their implicationin mate choice in the gregarious cockroach, Blattella germanica(L.). Binary choice tests showed that females mated preferentiallywith nonsibling rather than with sibling males, thus avoidingincest. In addition, inbreeding induced an important decreaseof their reproductive success. Contrary to what could be expectedwhen females had the choice between a nonsibling strain memberand a nonstrain member, they did not avoid mating with distantlyrelated nonstrain members, and extreme outbreeding induced anincrease of their reproductive success. Furthermore, our matechoice experiments disentangled the influences of familiarityfrom those of relatedness and evidenced that kin discriminationwas based on genetic cues independently of familiarity. Phenotypematching was a plausible mechanism for kin recognition. Contraryto many insect species, body size was not a salient criterionfor mate choice and had no consequences on reproductive success.     

Synthesis of phosphorylated recognition marker in lysosomal enzymes is located in the cis part of Golgi apparatus

Rat liver membranes were subjected to centrifugation in a sucrose density gradient in which the Golgi apparatus was separated into several subfractions. Two enzymes involved in the synthesis of the phosphorylated recognition marker in lysosomal enzymes, UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase fractionated with alpha-1,2-mannosidase, a marker enzyme of cis Golgi membranes and differently from galactosyltransferase, a marker enzyme of trans Golgi membranes.