bluuebunny/crossref_metadata_2025 huggingface dataset:
The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus.
Authors: Sakaguchi T , Leser G P , Lamb R A
Published: May 2004

High level expression of the M2 ion channel protein of influenza virus inhibits the rate of intracellular transport of the influenza virus hemagglutinin (HA) and that of other integral membrane glycoproteins. HA coexpressed with M2 is properly folded, is not associated with GRP78-BiP, and trimerizes with the same kinetics as when HA is expressed alone. Analysis of the rate of transport of HA from the ER to the cis and medial golgi compartments and the TGN indicated that transport through the Golgi apparatus is delayed. Uncleaved HA0 was not expressed at the cell surface, and accumulation HA at the plasma membrane was reduced to 75-80% of control cells. The delay in intracellular transport of HA on coexpression of M2 was not observed in the presence of the M2-specific ion channel blocker, amantadine, indicating that the Golgi transport delay is due to the M2 protein ion channel activity equilibrating pH between the Golgi lumen and the cytoplasm, and not due to saturation of the intracellular transport machinery. The Na+/H+ ionophore, monensin, which also equilibrates pH between the Golgi lumen and the cytoplasm, caused a similar inhibition of intracellular transport as M2 protein expression did for HA and other integral membrane glycoproteins. EM data showed a dilation of Golgi cisternae in cells expressing the M2 ion channel protein. Taken together, the data suggest a similarity of effects of M2 ion channel activity and monensin on intracellular transport through the Golgi apparatus.
A novel adaptor-related protein complex.
Authors: Simpson F , Bright N A , West M A , Newman L S , Darnell R B , Robinson M S
Published: May 2004

Coat proteins are required for the budding of the transport vesicles that mediate membrane traffic pathways, but for many pathways such proteins pathways, but for many pathways such proteins have not yet been identified. We have raised antibodies against p47, a homologue of the medium chains of the adaptor complexes of clathrin-coated vesicles (Pevsner, J., W. Volknandt, B.R. Wong, and R.H. Scheller. 1994. Gene (Amst.). 146:279-283), to determine whether this protein might be a component of a new type of coat. p47 coimmunoprecipitates with three other proteins: two unknown proteins of 160 and 25 kD, and beta-NAP, a homologue of the beta/beta'-adaptins, indicating that it is a subunit of an adaptor-like heterotetrameric complex. However, p47 is not enriched in preparations of clathrin-coated vesicles. Recruitment of the p47-containing complex onto cell membranes is stimulated by GTP gamma S and blocked by brefeldin A, indicating that, like other coat proteins, its membrane association is regulated by an ARF. The newly recruited complex is localized to non-clathrin-coated buds and vesicles associated with the TGN. Endogenous complex in primary cultures of neuronal cells is also localized to the TGN, and in addition, some complex is associated with the plasma membrane. These results indicate that the complex is a component of a novel type of coat that facilitates the budding of vesicles from the TGN, possibly for transporting newly synthesized proteins to the plasma membrane.
Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.
Authors: Majoul I V , Bastiaens P I , Söling H D
Published: May 2004

The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3',5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.
Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases.
Authors: Deckert M , Ticchioni M , Bernard A
Published: May 2004

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells.
The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein, PKD1.
Authors: Moy G W , Mendoza L M , Schulz J R , Swanson W J , Glabe C G , Vacquier V D
Published: May 2004

During fertilization, the sea urchin sperm acrosome reaction (AR), an ion channel-regulated event, is triggered by glycoproteins in egg jelly (EJ). A 210-kD sperm membrane glycoprotein is the receptor for EJ (REJ). This conclusion is based on the following data: purified REJ binds species specifically to EJ dotted onto nitrocellulose, an mAb to REJ induces the sperm AR, antibody induction is blocked by purified REJ, and purified REJ absorbs the AR-inducing activity of EJ. Overlapping fragments of REJ cDNA were cloned (total length, 5,596 bp). The sequence was confirmed by microsequencing six peptides of mature REJ and by Western blotting with antibody to a synthetic peptide designed from the sequence. Complete deglycosylation of REJ followed by Western blotting yielded a size estimate in agreement with that of the mature amino acid sequence. REJ is modular in design; it contains one EGF module and two C-type lectin carbohydrate-recognition modules. Most importantly, it contains a novel module, herein named the REJ module (700 residues), which shares extensive homology with the human polycystic kidney disease protein (PKD1). Mutations in PKD1 cause autosomal dominant polycystic kidney disease, one of the most frequent genetic disease of humans. The lesion in cellular physiology resulting from mutations in the PKD1 protein remains unknown. The homology between REJ modules of the sea urchin REJ and human PKD1 suggests that PKD1 could be involved in ionic regulation.
Distinct cellular and subcellular patterns of expression imply distinct functions for the Drosophila homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin.
Authors: McCartney B M , Fehon R G
Published: May 2004

Interest in members of the protein 4.1 super-family, which includes the ezrin-radixin-moesin (ERM) group, has been stimulated recently by the discovery that the human neurofibromatosis 2 (NF2) tumor suppressor gene encodes an ERM-like protein, merlin. Although many proteins in this family are thought to act by linking the actin-based cytoskeleton to transmembrane proteins, the cellular functions of merlin have not been defined. To investigate the cellular and developmental functions of these proteins, we have identified and characterized Drosophila homologues of moesin (Dmoesin) and of the NF2 tumor suppressor merlin (Dmerlin). Using specific antibodies, we show that although these proteins are frequently coexpressed in developing tissues, they display distinct subcellular localizations. While Dmoesin is observed in continuous association with the plasma membrane, as is typical for an ERM family protein, Dmerlin is found in punctuate structures at the membrane and in the cytoplasm. Investigation of Dmerlin cultured cells demonstrates that it is associated with endocytic compartments. As a result of these studies, we propose that the merlin protein has unique functions in the cell which differ from those of other ERM family members.
Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice.
Authors: Galou M , Colucci-Guyon E , Ensergueix D , Ridet J L , Gimenez y Ribotta M , Privat A , Babinet C , Dupouey P
Published: May 2004

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed predominantly in astrocytes. The study of its expression in the astrocyte lineage during development and in reactive astrocytes has revealed an intricate relationship with the expression of vimentin, another intermediate filament protein widely expressed in embryonic development. these findings suggested that vimentin could be implicated in the organization of the GFAP network. To address this question, we have examined GFAP expression and network formation in the recently generated vimentin knockout (Vim-) mice. We show that the GFAP network is disrupted in astrocytes that normally coexpress vimentin and GFAP, e.g., those of the corpus callosum or the Bergmann glia of cerebellum. Furthermore, Western blot analysis of GFAP protein content in the cerebellum suggests that posttranslational mechanisms are implicated in the disturbance of GFAP network formation. The role of vimentin in this process was further suggested by transfection of Vim-cultured astrocytes with a vimentin cDNA, which resulted in the normal assembly of the GFAP network. Finally, we examined GFAP expression after stab wound-induced astrogliosis. We demonstrate that in Vim- mice, reactive astrocytes that normally express both GFAP and vimentin do not exhibit GFAP immunoreactivity, whereas those that normally express GFAP only retain GFAP immunoreactivity. Taken together, these results show that in astrocytes, where vimentin is normally expressed with GFAP fails to assemble into a filamentous network in the absence of vimentin. In these cells, therefore, vimentin appears necessary to stabilize GFAP filaments and consequently the network formation.
The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells.
Authors: Knibbs R N , Craig R A , Natsuka S , Chang A , Cameron M , Lowe J B , Stoolman L M
Published: May 2004

Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere. This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E-selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated Lewis(x)-related sLe(x)related epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state levels FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLe(x)-related epitopes and the synthesis of E-selectin ligands which equal or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe(x,a) epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis(x) structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts. Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT-VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis. FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none. We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells.
Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death.
Authors: Jacobsen M D , Weil M , Raff M C
Published: May 2004

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.
Constitutive expression of the machinery for programmed cell death.
Authors: Weil M , Jacobson M D , Coles H S , Davies T J , Gardner R L , Raff K D , Raff M C
Published: May 2004

In the presence of cycloheximide (CHX) to inhibit protein synthesis, a high concentration of staurosporine (STS) induces almost all cells in explant cultures of 8/8 types of newborn mouse organs and 3/3 types of adult mouse organs to die with the characteristic features of apoptosis. Eggs and blastomeres also die in this way when treated with STS and CHX, although they are less sensitive to this treatment than trophectoderm or inner cell mass cells whose sensitivity resembles that of other developing cells. Human red blood cells are exceptional in being completely resistant to treatment with STS and CHX. As (STS plus CHX)-induced cell deaths have been shown to display the characteristic features of programmed cell death (PCD), we conclude that all mammalian nucleated cells are capable of undergoing PCD and constitutively express all the proteins required to do so. It seems that the machinery for PCD is in place and ready to run, even though its activation often depends on new RNA and protein synthesis.
Subunit composition of neurofilaments specifies axonal diameter.
Authors: Xu Z , Marszalek J R , Lee M K , Wong P C , Folmer J , Crawford T O , Hsieh S T , Griffin J W , Cleveland D W
Published: May 2004

Neurofilaments (NFs), which are composed of NF-L, NF-M, and NF-H, are required for the development of normal axonal caliber, a property that in turn is a critical determinant of axonal conduction velocity. To investigate how each subunit contributes to the radial growth of axons, we used transgenic mice to alter the subunit composition of NFs. Increasing each NF subunit individually inhibits radial axonal growth, while increasing both NF-M and NF-H reduces growth even more severely. An increase in NF-L results in an increased filament number but reduced interfilament distance. Conversely, increasing NF-M, NF-H, or both reduces filament number, but does not alter nearest neighbor interfilament distance. Only a combined increase of NF-L with either NF-M or NF-H promotes radial axonal growth. These results demonstrate that both NF-M and NF-H play complementary roles with NF-L in determining normal axonal calibers.
Sequence and transmembrane topology of MEC-4, an ion channel subunit required for mechanotransduction in Caenorhabditis elegans.
Authors: Lai C C , Hong K , Kinnell M , Chalfie M , Driscoll M
Published: May 2004

The process by which mechanical stimuli are converted into cellular responses is poorly understood, in part because key molecules in this mode of signal transduction, the mechanically gated ion channels, have eluded cloning efforts. The Caenorhabditis elegans mec-4 gene encodes a subunit of a candidate mechanosensitive ion channel that plays a critical role in touch reception. Comparative sequence analysis of C. elegans and Caenorhabditis briggsae mec-4 genes was used to initiate molecular studies that establish MEC-4 as a 768-amino acid protein that includes two hydrophobic domains theoretically capable of spanning a lipid bilayer. Immunoprecipitation of in vitro translated mec-4 protein with domain-specific anti-MEC-4 antibodies and in vivo characterization of a series of mec-4lacZ fusion proteins both support the hypothesis that MEC-4 crosses the membrane twice. The MEC-4 amino- and carboxy-terminal domains are situated in the cytoplasm and a large domain, which includes three Cys-rich regions, is extracellular. Definition of transmembrane topology defines regions that might interact with the extracellular matrix or cytoskeleton to mediate mechanical signaling.
Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression.
Authors: Kretz-Remy C , Mehlen P , Mirault M E , Arrigo A P
Published: May 2004

We report here that both kappa B-dependent transactivation of a reporter gene and NF-kappa B activation in response to tumor necrosis factor (TNF alpha) or H2O2 treatments are deficient in human T47D cell transfectants that overexpress seleno-glutathione peroxidase (GSHPx). These cells feature low reactive oxygen species (ROS) levels and decreased intracellular ROS burst in response to TNF alpha treatment. Decreased ROS levels and NF-kappa B activation were likely to result from GSHPx increment since these phenomena were no longer observed when GSHPx activity was reduced by selenium depletion. The cellular contents of the two NF-kappa B subunits (p65 and p50) and of the inhibitory subunit I kappa B-alpha were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of Nf-kappa B. Nuclear translocation of NF-kappa B as well as I kappa B-alpha degradation were inhabited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNF alpha, a time correlation was observed between elevated ROS levels and I kappa B-alpha degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of I kappa B-alpha, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNF alpha-mediated transient accumulation of the acidic and highly phosphorylated I kappa B-alpha isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of I kappa B-alpha, a phenomenon that precedes and controls the degradation of this protein, and then NF-kappa B activation.
Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis.
Authors: Cardone M H , Smith B L , Mennitt P A , Mochly-Rosen D , Silver R B , Mostov K E
Published: May 2004

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5-trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.
The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear pore complex.
Authors: Nehrbass U , Rout M P , Maguire S , Blobel G , Wozniak R W
Published: May 2004

We have isolated a major protein constituent from a highly enriched fraction of yeast nuclear pore complexes (NPCs). The gene encoding this protein, Nup188p, was cloned, sequenced, and found to be nonessential upon deletion. Nup188p cofractionates with yeast NPCs and gives an immunofluorescent staining pattern typical of nucleoporins. Using immunoelectron microscopy, Nup188p was shown to localize to both the cytoplasmic and nucleoplasmic faces of the NPC core. There, Nup188p interacts with an integral protein of the pore membrane domain, Pom152p, and another abundant nucleoporin, Nic96p. The effects of various mutations in the NUP188 gene on the structure of the nuclear envelope and the function of the NPC were examined. While null mutants of NUP188 appear normal, other mutants allelic to NUP188 exhibit a dominant effect leading to the formation of NPC-associated nuclear envelope herniations and growth inhibition at 37 degrees C. In addition, depletion of the interacting protein Pom152p in cells lacking Nup188p resulted in severe deformations of the nuclear envelope. We suggest that Nup188p is one of a group of proteins that form the octagonal core structure of the NPC and thus functions in the structural organization of the NPC and nuclear envelope.
A biosynthetic regulated secretory pathway in constitutive secretory cells.
Authors: Chavez R A , Miller S G , Moore H P
Published: May 2004

It has frequently been proposed that while the constitutive secretory pathway is present in all cells, the regulated secretory pathway is found only in specialized cells such as neuronal, endocrine, or exocrine types. In this study we provide evidence that suggests that this distinction is not as restrictive as proposed. We have identified a population of post-Golgi storage vesicles in several constitutive secretory cells using [35S]SO4-labeled glycosaminoglycan (GAG) chains as a marker. A fraction of this pool of vesicles can undergo exocytosis in response to stimuli such as cytoplasmic Ca2+ and phorbol esters. The effect of Ca2+ was demonstrated both in intact cells in the presence of the ionophore A23187 and in streptolysin-O-permeabilized semi-intact cells. N-ethylmaleiimide, under conditions known to block regulated and constitutive secretion, inhibited the stimulated secretion from these cells, suggesting that the observed release of labeled GAG chains was not due to a leakage artefact. Subcellular fractionation revealed that the stored GAG chains were in low-density membrane granules (d approximately 1.12 g/ml), whose size was greater than that of synaptic-like vesicles found in PC12 cells. In addition, in CHO cells that express epitope-tagged rab 3D, the labeled GAG chains were found to cofractionate with the exogenous rab protein. When expressed in the regulated cell line AtT-20, this tagged rab protein was found to colocalize with ACTH-containing dense-core granules by indirect immunofluorescence. Taken together, these results provide evidence for the presence of a cryptic regulated secretory pathway in "constitutive" cells and suggest that the regulated secretory pathway is more widespread amongst different cell types than previously believed.
Oligomerization and maturation of Na,K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit.
Authors: Geering K , Beggah A , Good P , Girardet S , Roy S , Schaer D , Jaunin P
Published: May 2004

Subunit assembly plays an essential role in the maturation of oligomeric proteins. In this study, we have characterized the main structural and functional consequences of the assembly of alpha and beta subunits of Na,K-ATPase. Xenopus oocytes injected with alpha and/or beta cRNA were treated with brefeldin A, which permitted the accumulation of individual subunits or alpha-beta complexes in the ER. Only alpha subunits that are associated with beta subunits become resistant to trypsin digestion and cellular degradation. Similarly, assembly with beta subunits is necessary and probably sufficient for the catalytic alpha subunit to acquire its main functional properties at the level of the ER, namely the ability to adopt different ligand-dependent conformations and to hydrolyze ATP in an Na(+)- and K(+)-dependent, ouabain-inhibitable fashion. Not only the alpha but also the beta subunit undergoes a structural change after assembly, which results in a global increase in its protease resistance. Furthermore, extensive and controlled proteolysis assays on wild-type and NH2-terminally modified beta subunits revealed a K(+)-dependent interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit, which is likely to be involved in the modulation of the K(+)-activation of the Na,K-pump transport activity. Thus, we conclude that the ER assembly process not only establishes the basic structural interactions between individual subunits, which are required for the maturation of oligomeric proteins, but also distinct, functional interactions, which are involved in the regulation of functional properties of mature proteins.
A novel suppressor of ras1 in fission yeast, byr4, is a dosage-dependent inhibitor of cytokinesis.
Authors: Song K , Mach K E , Chen C Y , Reynolds T , Albright C F
Published: May 2004

A novel gene, designated byr4, was identified in Schizosaccharomyces pombe that affects the mitotic cell cycle and shows genetic interactions with the ras1 signaling pathways. Null alleles of byr4 cause cell cycle arrest in late mitosis and permit multiple rounds of septation. The multiple septa typically divide two nuclei, but the nuclei frequently do not stain equally with 4',6-diamidino-2-phenylindole (DAPI), suggesting that byr4 is required for proper karyokinesis. Overexpression of byr4 inhibits cytokinesis, but cell cycle progression continues leading to multinucleate cells. When byr4 is overexpressed, the early steps in the cytokinesis pathway, including formation of the medial F-actin ring, occur normally; however, the later steps in the pathway, including contraction of the F-actin ring, septation, and rearrangement of the medial F-actin following mitosis, rarely occur, byr4 shows two genetic interactions with ras1. The inhibition of cytokinesis by byr4 overexpression was exacerbated by null alleles of ras1 and scd1, suggesting a link between pathways needed for cell polarity and cytokinesis. Overexpression of byr4 also partially bypasses the need for ras1 for sporulation. The electrophoretic mobility of the byr4 protein varied in response to mutants that perturb cytokinesis and karyokinesis, suggesting interactions between byr4 and these gene products. A more rapidly migrating byr4 protein was found in cells with mutations in cdc16, which undergo repeated septation, and in cdc15, which fail to form a medial F-actin ring in mitosis. A slower migrating byr4 protein was found in cells with a mutation in the beta-tubulin gene, which arrests cells at the metaphase-anaphase transition.
armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila.
Authors: Müller H A , Wieschaus E
Published: May 2004

Cellularization of the Drosophila embryo results in the formation of a cell monolayer with many characteristics of a polarized epithelium. We have used antibodies specific to cellular junctions and nascent plasma membranes to study the formation of the zonula adherens (ZA) in relation to the establishment of basolateral membrane polarity. The same approach was then used as a test system to identify X-linked zygotically active genes required for ZA formation. We show that ZA formation begins during cellularization and that the basolateral membrane domain is established at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome, we have identified genes that are required for the formation of the ZA and the generation of basolateral membrane polarity. We show that embryos mutant for both stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to establish the ZA, the formation of the monolayered epithelium is disrupted after cellularization, resulting in formation of a multilayered cell sheet by mid-gastrulation. SEM analysis of mutant embryos revealed a conversion of cells exhibiting epithelial characteristics into cells exhibiting mesenchymal characteristics. To investigate how mutations that affect an integral component of the ZA itself influence ZA formation, we examined embryos with reduced maternal and zygotic supply of wild-type Arm protein. These embryos, like embryos mutant for both sdt and baz, exhibit an early disruption of ZA formation. These results suggest that early stages in the assembly of the ZA are critical for the stability of the polarized blastoderm epithelium.
Biology of chemokine and classical chemoattractant receptors: differential requirements for adhesion-triggering versus chemotactic responses in lymphoid cells.
Authors: Campbell J J , Qin S , Bacon K B , Mackay C R , Butcher E C
Published: May 2004

Several chemoattractant receptors can support agonist-induced, integrin-dependent arrest of rolling neutrophils in inflamed venules in vivo, as well as subsequent crawling into tissues. It has been hypothesized that receptors of the Galpha(i)-linked chemoattractant subfamilies, especially receptors for chemokines, may mediate parallel activation-dependent arrest of homing lymphocyte subsets. However, although several chemokines can attract subsets of B or T cells, robust chemoattractant triggering of resting lymphocyte adhesion to vascular ligands has not been observed. To study the biology of individual leukocyte chemoattractant receptors in a defined lymphoid environment, mouse L1/2 pre-B cells and/or human Jurkat T cells were transfected with alpha (IL-8 receptor A) or beta (MIP-1alpha/CC-CKR-1) chemokine receptors, or with the classical chemoattractant C5a (C5aR) or formyl peptide receptors (fPR). All receptors supported robust agonist-dependent alpha4beta1 integrin-mediated adhesion of lymphocytes to VCAM-1. L1/2 cells cotransfected with fPR and beta7 integrin were also induced to bind MAdCAM-1, suggesting common mechanisms coupling chemoattractant receptors to activation of distinct integrins. Adhesion was rapid but transient, with spontaneous reversion to unstimulated levels within 5 min after peak binding. When observed under flow conditions, alpha4beta1-mediated arrest occurred within seconds after initiation of contact and rolling of IL-8RA transfectants on VCAM-1/IL-8 co-coated surface; and arrest reversed spontaneously after a mean of 5 min with a return to rolling behavior. Each of the receptors also conferred agonist-specific chemotaxis; however, whereas strong adhesion required simultaneous occupancy of many receptors with maximal responses above the Kd, chemotaxis in each case was suppressed at high agonist concentrations. The findings indicate that alpha and beta chemokine as well as classical chemoattractant receptors can trigger robust adhesion as well as directed migration of lymphoid cells, but that the requirements for and kinetics of adhesion triggering and chemotaxis are distinct, thus permitting their independent regulation. They suggest that the discordance between proadhesive and chemoattractant responses of circulating lymphocytes to many chemokines may reflect quantitative aspects of receptor expression and/or coupling rather than qualitative differences in receptor signaling.
The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme.
Authors: Sakai Y , Saiganji A , Yurimoto H , Takabe K , Saiki H , Kato N
Published: May 2004

Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C. boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. Boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed. Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol. Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases. Methanol-induced cells of pmp47delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes. Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus. We disrupted PMP47 in these peroxisome-deficient mutants. In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus. We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes. Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains. Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.
Drosophila paramyosin/miniparamyosin gene products show a large diversity in quantity, localization, and isoform pattern: a possible role in muscle maturation and function.
Authors: Maroto M , Arredondo J , Goulding D , Marco R , Bullard B , Cervera M
Published: May 2004

The Drosophila paramyosin/miniparamyosin gene expresses two products of different molecular weight transcriptionally regulated from two different promoters. Distinct muscle types also have different relative amounts of myosin, paramyosin, and miniparamyosin, reflecting differences in the organization of their thick filaments. Immunofluorescence and EM data indicate that miniparamyosin is mainly located in the M line and at both ends of the thick filaments in Drosophila indirect flight muscles, while paramyosin is present all along the thick filaments. In the tergal depressor of the trochanter muscle, both proteins are distributed all along the A band. In contrast, in the waterbug, Lethocerus, both paramyosin and miniparamyosin are distributed along the length of the indirect flight and leg muscle thick filaments. Two-dimensional and one-dimensional Western blot analyses have revealed that miniparamyosin has several isoforms, focusing over a very wide pH range, all of which are phosphorylated in vivo. The changes in isoform patterns of miniparamyosin and paramyosin indicate a direct or indirect involvement of these proteins in muscle function and flight. This wide spectrum of potential regulatory characteristics underlines the key importance of paramyosin/miniparamyosin and its complex isoform pattern in the organization of the invertebrate thick filament.
The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery.
Authors: McBride H M , Goping I S , Shore G C
Published: May 2004

Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal-anchor sequence. The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment. The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human). Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed. This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts. The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins. In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein-translocation machinery. These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide.
Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.
Authors: Ma Y , Kouranov A , LaSala S E , Schnell D J
Published: May 2004

The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.
Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.
Authors: Vogel S S , Blank P S , Zimmerberg J
Published: May 2004

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.
Host cell invasion by trypanosomes requires lysosomes and microtubule/kinesin-mediated transport.
Authors: Rodríguez A , Samoff E , Rioult M G , Chung A , Andrews N W
Published: May 2004

Invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi occurs by an actin-independent mechanism distinct from phagocytosis. Clusters of host lysosomes are observed at the site of parasite attachment, and lysosomal markers are detected in the vacuolar membrane at early stages of the entry process. These observations led to the hypothesis that the trypanosomes recruit host lysosomes to their attachment site, and that lysosomal fusion serves as a source of membrane to form the parasitophorous vacuole. Here we directly demonstrate directional migration of lysosomes to the parasite entry site, using time-lapse video-enhanced microscopy of L6E9 myoblasts exposed to T. cruzi trypomastigotes. BSA-gold-loaded lysosomes moved towards the cell periphery, in the direction of the parasite attachment site, but only when their original position was less than 11-12 microns from the invasion site. Lysosomes more distant from the invasion area exhibited only the short multi-directional saltatory movements previously described for lysosomes, regardless of their proximity to the cell margins. Specific depletion of peripheral lysosomes was obtained by microinjection of NRK cells with antibodies against the cytoplasmic domain of lgp 120, a treatment that aggregated lysosomes in the perinuclear area and inhibited T. cruzi entry. The microtubule-binding drugs nocodazole, colchicine, vinblastine, and taxol also inhibited invasion, in both NRK and L6E9 cells. Furthermore, microinjection of antibodies to the heavy chain of kinesin blocked the acidification-induced, microtubule-dependent redistribution of lysosomes to the host cell periphery, and reduced trypomastigote entry. Our results therefore demonstrate that during T. cruzi invasion of host cells lysosomes are mobilized from the immediately surrounding area, and that availability of lysosomes at the cell periphery and microtubule/kinesin-mediated transport are requirements for parasite entry.
Sphingolipids and glycoproteins are differentially trafficked to the Chlamydia trachomatis inclusion.
Authors: Scidmore M A , Fischer E R , Hackstadt T
Published: May 2004

Chlamydia trachomatis is an obligate intracellular pathogen that multiples within the confines of a membrane-bound vacuole called an inclusion. Approximately 40-50% of the sphingomyelin synthesized from exogenously added NBD-ceramide is specifically transported from the Golgi apparatus to the chlamydial inclusion (Hackstadt, T., M.A. Scidmore, and D.D. Rockey. 1995. Proc. Natl. Acad. Sci. USA. 92: 4877-4881). Given this major disruption of a cellular exocytic pathway and the similarities between glycolipid and glycoprotein exocytosis, we wished to determine whether the processing and trafficking of glycoproteins through the Golgi apparatus to the plasma membrane in chlamydia-infected cells was also disrupted. We analyzed the processing of several model glycoproteins including vesicular stomatitis virus G-protein, transferrin receptor, and human histocompatibility leukocyte class I antigen. In infected cells, the posttranslational processing and trafficking of these specific proteins through the Golgi apparatus and subsequent transport to the plasma membrane was not significantly impaired, nor were these glycoproteins found associated with the chlamydial inclusion membrane. Studies of receptor recycling from endocytic vesicles employing fluorescently and HRP-tagged transferrin and anti-transferrin receptor antibody revealed an increased local concentration of transferrin and transferrin receptor around but never within the chlamydial inclusion. However, Scatchard analysis failed to show either an increased intracellular accumulation of transferrin receptor or a decreased number of plasma membrane receptors in infected cells. Furthermore, the rate of exocytosis from the recycling endosomes to the plasma membrane was not altered in chlamydia-infected cells. Thus, although C. trachomatis disrupts the exocytosis of sphingolipids and the Golgi apparatus appears physically distorted, glycosylation and exocytosis of representative secreted and endocytosed proteins are not disrupted. These results suggest the existence of a previously unrecognized sorting of sphingolipids and glycoproteins in C. trachomatis-infected cells.