Localization and Recycling of gp27 (hp24γ3): Complex Formation with Other p24 Family Members

We report here the characterization of gp27 (hp24γ3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15°C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24α2), p24 (hp24β1), and p23 (hp24δ1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24γ4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.

Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

Differential Distribution of Dynamin Isoforms in Mammalian Cells

Dynamins are 100-kDa GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (RT)-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for dynamin I and nine for dynamin III. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of six different spliced forms tagged with green fluorescent protein. Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.

Interaction of Coatomer with Aminoglycoside Antibiotics: Evidence That Coatomer Has at Least Two Dilysine Binding Sites

Coatomer is the soluble precursor of the COPI coat (coat protein I) involved in traffic among membranes of the endoplasmic reticulum and the Golgi apparatus. We report herein that neomycin precipitates coatomer from cell extracts and from purified coatomer preparations. Precipitation first increased and then decreased as the neomycin concentration increased, analogous to the precipitation of a polyvalent antigen by divalent antibodies. This suggested that neomycin cross-linked coatomer into large aggregates and implies that coatomer has two or more binding sites for neomycin. A variety of other aminoglycoside antibiotics precipitated coatomer, or if they did not precipitate, they interfered with the ability of neomycin to precipitate. Coatomer is known to interact with a motif (KKXX) containing adjacent lysine residues at the carboxyl terminus of the cytoplasmic domains of some membrane proteins resident in the endoplasmic reticulum. All of the antibiotics that interacted with coatomer contain at least two close amino groups, suggesting that the antibiotics might be interacting with the di-lysine binding site of coatomer. Consistent with this idea, di-lysine itself blocked the interaction of antibiotics with coatomer. Moreover, di-lysine and antibiotics each blocked the coating of Golgi membranes by coatomer. These data suggest that certain aminoglycoside antibiotics interact with di-lysine binding sites on coatomer and that coatomer contains at least two of these di-lysine binding sites.

Dopamine tone regulates D1 receptor trafficking and delivery in striatal neurons in dopamine transporter-deficient mice

In vivo, G protein-coupled receptors (GPCR) for neurotransmitters undergo complex intracellular trafficking that contribute to regulate their abundance at the cell surface. Here, we report a previously undescribed alteration in the subcellular localization of D1 dopamine receptor (D1R) that occurs in vivo in striatal dopaminoceptive neurons in response to chronic and constitutive hyperdopaminergia. Indeed, in mice lacking the dopamine transporter, D1R is in abnormally low abundance at the plasma membrane of cell bodies and dendrites and is largely accumulated in rough endoplasmic reticulum and Golgi apparatus. Decrease of striatal extracellular dopamine concentration with 6-hydroxydopamine (6- OHDA) in heterozygous mice restores delivery of the receptor from the cytoplasm to the plasma membrane in cell bodies. These results demonstrate that, in vivo, in the central nervous system, the storage in cytoplasmic compartments involved in synthesis and the membrane delivery contribute to regulate GPCR availability and abundance at the surface of the neurons under control of the neurotransmitter tone. Such regulation may contribute to modulate receptivity of neurons to their endogenous ligands and related exogenous drugs.

Localization of α1,3-fucosyltransferase VI in Weibel–Palade bodies of human endothelial cells

Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked α1→3 to N-acetylglucosamine. A family of α1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription–PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with β4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel–Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel–Palade bodies suggests a function independent of Golgi-associated glycosylation.

Galactose-extended glycans of antibodies produced by transgenic plants

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human β1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal β1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal β1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human β1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia.

Arabidopsis Sec21p and Sec23p Homologs. Probable Coat Proteins of Plant COP-Coated Vesicles1

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.

Characterization of Chlamydomonas reinhardtii Zygote-Specific cDNAs That Encode Novel Proteins Containing Ankyrin Repeats and WW Domains1

Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3 mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles.

Intracellular Localization of Phospholipase D1 in Mammalian Cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.

Transport of Sterols to the Plasma Membrane of Leek Seedlings1

To investigate the intracellular transport of sterols in etiolated leek (Allium porrum L.) seedlings, in vivo pulse-chase experiments with [1-14C]acetate were performed. Then, endoplasmic reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were prepared and analyzed for the radioactivity incorporated into free sterols. In leek seedlings sterols are present as a mixture in which (24R)-24-ethylcholest-5-en-3β-ol is by far the major compound (around 60%). The other sterols are represented by cholest-5-en-3β-ol, 24-methyl-cholest-5-en-3β-ol, (24S)-24-ethylcholesta-5,22E-dien-3β-ol, and stigmasta-5,24(241)Z-dien-3β-ol. These compounds are shown to reside mainly in the PM. Our results clearly indicate that free sterols are actively transported from the endoplasmic reticulum to the PM during the first 60 min of chase, with kinetics very similar to that of phosphatidylserine. Such a transport was found to be decreased at low temperature (12°C) and following treatment with monensin and brefeldin A. These data are consistent with a membrane-mediated process for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus.

Transdifferentiation of Mature Cortical Cells to Functional Abscission Cells in Bean1

Abscission explants of bean (Phaseolus vulgaris L.) were treated with ethylene to induce cell separation at the primary abscission zone. After several days of further incubation of the remaining petiole in endogenously produced ethylene, the distal two-thirds of the petiole became senescent, and the remaining (proximal) portion stayed green. Cell-to-cell separation (secondary abscission) takes place precisely at the interface between the senescing yellow and the enlarging green cells. The expression of the abscission-associated isoform of β-1,4-glucanhydrolase, the activation of the Golgi apparatus, and enhanced vesicle formation occurred only in the enlarging cortical cells on the green side. These changes were indistinguishable from those that occur in normal abscission cells and confirm the conversion of the cortical cells to abscission-type cells. Secondary abscission cells were also induced by applying auxin to the exposed primary abscission surface after the pulvinus was shed, provided ethylene was added. Then, the orientation of development of green and yellow tissue was reversed; the distal tissue remained green and the proximal tissue yellowed. Nevertheless, separation still occurred at the junction between green and yellow cells and, again, it was one to two cell layers of the green side that enlarged and separated from their senescing neighbors. Evaluation of Feulgen-stained tissue establishes that, although nuclear changes occur, the conversion of the cortical cell to an abscission zone cell is a true transdifferentiation event, occurring in the absence of cell division.

Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.

Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. Binding of cholera toxin to the cell surface and transport of toxin to the Golgi apparatus were not changed in thapsigargin-treated cells, suggesting increased retrograde transport of cholera toxin from the Golgi apparatus to the ER. The data demonstrate that retrograde transport of cholera toxin can take place and that the transport is under regulation. The results are consistent with the idea that retrograde transport can be important for the action of cholera toxin.

Sphingolipid synthesis as a target for chemotherapy against malaria parasites.

The human malaria parasite Plasmodium falciparum contains sphingomyelin synthase in its Golgi apparatus and in a network of tubovesicular membranes in the cytoplasm of the infected erythrocyte. Palmitoyl and decanoyl analogues of 1-phenyl-2-acylamino-3-morpholino-1-propanol inhibit the enzyme activity in infected erythrocytes. An average of 35% of the activity is extremely sensitive to these drugs and undergoes a rapid, linear decrease at drug concentrations of 0.05-1 microM. The remaining 65% suffers a slower linear inhibition at drug concentrations ranging from 25 to 500 microM. Evidence is presented that inhibition of the sensitive fraction alone selectively disrupts the appearance of the interconnected tubular network in the host cell cytoplasm, without blocking secretory development at the parasite plasma membrane or in organelles within the parasite, such as the Golgi and the digestive food vacuole. This inhibition also blocks parasite proliferation in culture, indicating that the sensitive sphingomyelin synthase activity as well as the tubovesicular network may provide rational targets for drugs against malaria.