Nucleotide Triphosphates Are Required for the Transport of Glycolate Oxidase into Peroxisomes1

All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.

Distribution and Transport of Cholesterol in Caenorhabditis elegans

Cholesterol transport is an essential process in all multicellular organisms. In this study we applied two recently developed approaches to investigate the distribution and molecular mechanisms of cholesterol transport in Caenorhabditis elegans. The distribution of cholesterol in living worms was studied by imaging its fluorescent analog, dehydroergosterol, which we applied to the animals by feeding. Dehydroergosterol accumulates primarily in the pharynx, nerve ring, excretory gland cell, and gut of L1–L3 larvae. Later, the bulk of dehydroergosterol accumulates in oocytes and spermatozoa. Males display exceptionally strong labeling of spermatids, which suggests a possible role for cholesterol in sperm development. In a complementary approach, we used a photoactivatable cholesterol analog to identify cholesterol-binding proteins in C. elegans. Three major and several minor proteins were found specifically cross-linked to photocholesterol after UV irradiation. The major proteins were identified as vitellogenins. rme-2 mutants, which lack the vitellogenin receptor, fail to accumulate dehydroergosterol in oocytes and embryos and instead accumulate dehydroergosterol in the body cavity along with vitellogenin. Thus, uptake of cholesterol by C. elegans oocytes occurs via an endocytotic pathway involving yolk proteins. The pathway is a likely evolutionary ancestor of mammalian cholesterol transport.

An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine −3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the “ear” domain of the clathrin adaptor AP-1 γ subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Δ), the major Gga protein, accentuates growth and α-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Δ or a deletion of the AP-1 β subunit gene (apl2Δ) alone are phenotypically normal, but cells carrying both gga2Δ and apl2Δ are defective in growth, α-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.

Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight.

Since concomitant release of structurally related peptide hormones with apparently similar functions seems to be a general concept in endocrinology, we have studied the dynamics of the lifetime of the three known adipokinetic hormones (AKHs) of the migratory locust, which control flight-directed mobilization of carbohydrate and lipid from fat body stores. Although the structure of the first member of the AKHs has been known for 20 years, until now, reliable data on their inactivation and removal from the hemolymph are lacking, because measurement requires AKHs with high specific radioactivity. Employing tritiated AKHs with high specific radioactivity, obtained by catalytic reduction with tritium gas of the dehydroLeu2 analogues of the AKHs synthesized by the solid-phase procedure, studies with physiological doses of as low as 1.0 pmol per locust could be conducted. The AKHs appear to be transported in the hemolymph in their free forms and not associated with a carrier protein, despite their strong hydrophobicity. Application of AKHs in their free form in in vivo and in vitro studies therefore now has been justified. We have studied the degradation of the three AKHs during rest and flight. The first cleavage step by an endopeptidase is crucial, since the resulting degradation products lack any adipokinetic activity. Half-lives for AKH-I, -II and -III were 51, 40, and 5 min, respectively, for rest conditions and 35, 37, and 3 min, respectively, during flight. The rapid and differential degradation of structurally related hormones leads to changes in the ratio in which they are released and therefore will have important consequences for concerted hormone action at the level of the target organ or organs, suggesting that each of the known AKHs may play its own biological role in the overall syndrome of insect flight.

The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor.

More than 30 years ago, Brambell published the hypothesis bearing his name [Brambell, F. W. R., Hemmings, W. A. & Morris, 1. C. (1964) Nature (London) 203, 1352-1355] that remains as the cornerstone for thinking on IgG catabolism. To explain the long survival of IgG relative to other plasma proteins and its pattern of increased fractional catabolism with high concentrations of IgG, Brambell postulated specific IgG "protection receptors" (FcRp) that would bind IgG in pinocytic vacuoles and redirect its transport to the circulation; when the FcRp was saturated, the excess unbound IgG then would pass to unrestricted lysosomal catabolism. Brambell subsequently postulated the neonatal gut transport receptor (FcRn) and showed its similar saturable character. FcRn was recently cloned but FcRp has not been identified. Using a genetic knockout that disrupts the FcRn and intestinal IgG transport, we show that this lesion also disrupts the IgG protection receptor, supporting the identity of these two receptors. IgG catabolism was 10-fold faster and IgG levels were correspondingly lower in mutant than in wild-type mice, whereas IgA was the same between groups, demonstrating the specific effects on the IgG system. Disruption of the FcRp in the mutant mice was also shown to abrogate the classical pattern of decreased IgG survival with higher IgC concentration. Finally, studies in normal mice with monomeric antigen-antibody complexes showed differential catabolism in which antigen dissociates in the endosome and passes to the lysosome, whereas the associated antibody is returned to circulation; in mutant mice, differential catabolism was lost and the whole complex cleared at the same accelerated rate as albumin, showing the central role of the FcRp to the differential catabolism mechanism. Thus, the same receptor protein that mediates the function of the FcRn transiently in the neonate is shown to have its functionally dominant expression as the FcRp throughout life, resolving a longstanding mystery of the identity of the receptor for the protection of IgG. This result also identifies an important new member of the class of recycling surface receptors and enables the design of protein adaptations to exploit this mechanism to improve survivals of other therapeutic proteins in vivo.

cemA homologue essential to CO2 transport in the cyanobacterium Synechocystis PCC6803.

We have isolated mutants of Synechocystis PCC6803 that grew very slowly in a low-sodium medium, which is unfavorable for HCO3(-) transport, and examined two of these mutants (SC1 and SC2) for their ability to take up CO2 and HCO3(-) in the light. The CO2 transport activity of SC1 and SC2 was much lower than that of the wild type (WT), whereas there was no difference between the mutants and the WT in their activity of HCO3(-) transport. A clone containing a 3.9-kilobase-pair insert DNA that transforms both mutants to the WT phenotype was isolated from a genomic library of WT Synechocystis. Sequencing of the insert DNA in the region of mutations in SC1 and SC2 revealed an open reading frame (designated cotA), which showed significant amino-acid sequence homology to cemA encoding a protein found in the inner envelope membrane of chloroplasts. The cotA gene is present in a single copy and was not cotranscribed with any other gene(s). cotA encodes a protein of 247 amino acids containing four transmembrane domains. There was substitution of a single base in SC1 and two bases in SC2 in their cotA genes. A possible role of the cotA gene product in CO2 transport is discussed.

Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport.

An adipocyte membrane glycoprotein, (FAT), homologous to human CD36, has been previously implicated in the binding/transport of long-chain fatty acids. It bound reactive derivatives of long-chain fatty acids and binding was specific and associated with significant inhibition of fatty acid uptake. Tissue distribution of the protein and regulation of its expression were also consistent with its postulated role. In this report, we have examined the effects of FAT expression on rates and properties of fatty acid uptake by Ob17PY fibroblasts lacking the protein. Three clones (P21, P22, and P25) were selected based on FAT mRNA and protein levels. Cell surface labeling could be demonstrated with the anti-CD36 antibody FITC-OKM5. In line with this, the major fraction of immunoreactive FAT was associated with the plasma membrane fraction. Assays of oleate and/or palmitate uptake demonstrated higher rates in the three FAT-expressing clones, compared to cells transfected with the empty vector. Clone P21, which had the highest protein levels on Western blots, exhibited the largest increase in transport rates. Fatty acid uptake in FAT-expressing P21 cells reflected two components, a phloretin-sensitive high-affinity saturable component with a Km of 0.004 microM and a basal phloretin-insensitive component that was a linear function of unbound fatty acid. P21 cells incorporated more exogenous fatty acid into phospholipids, indicating that binding of fatty acids was followed by their transfer into the cell and that both processes were increased by FAT expression. The data support the interpretation that FAT/CD36 functions as a high-affinity membrane receptor/transporter for long-chain fatty acids.

Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation.

Cytoplasmic dynein is associated with slow axonal transport.

Neuronal function is dependent on the transport of materials from the cell body to the synapse via anterograde axonal transport. Anterograde axonal transport consists of several components that differ in both rate and protein composition. In fast transport, membranous organelles are moved along microtubules by the motor protein kinesin. The cytoskeleton and the cytomatrix proteins move in the two components of slow transport. While the mechanisms underlying slow transport are unknown, it has been hypothesized that the movement of microtubules in slow transport is generated by sliding. To determine whether dynein, a motor protein that causes microtubule sliding in flagella, may play a role in slow axonal transport, we identified the transport rate components with which cytoplasmic dynein is associated in rat optic nerve. Nearly 80% of the anterogradely moving dynein was associated with slow transport, whereas only approximately 15% of the dynein was associated with the membranous organelles of anterograde fast axonal transport. A segmental analysis of the transport of dynein through contiguous regions of the optic nerve and tract showed that dynein is associated with the microfilaments and other proteins of slow component b. Dynein from this transport component has the capacity to bind microtubules in vitro. These results are consistent with the hypothesis that cytoplasmic dynein generates the movement of microtubules in slow axonal transport. A model is presented to illustrate how dynein attached to the slow component b complex of proteins is appropriately positioned to generate force of the correct polarity to slide microtubules down the axon.

14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae.

Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.

An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.

Identifying the role of natural resources in knowledge-based strategies of development

Knowledge has adopted a preferential role in the explanation of development while the evidence about the effect of natural resources in countries’ performance is more controversial in the economic literature. This paper tries to demonstrate that natural resources may positively affect growth in countries with a strong natural resources specialization pattern although the magnitude of these effects depend on the type of resources and on other aspects related to the production and innovation systems. The positive trajectory described by a set of national economies mainly specialized in natural resources and low-tech industries invites us to analyze what is the combination of factors that serves as engine for a sustainable development process. With panel data for the period 1996-2008 we estimate an applied growth model where both traditional factors and other more related to innovation and absorptive capabilities are taken into account. Our empirical findings show that according to the postulates of a knowledge-based approach, a framework that combines physical and intangible factors is more suitable for the definition of development strategies in those prosperous economies dominated by natural resources and connected activities, while the internationalization process of activities and technologies become also a very relevant aspect.

Fostering Change: Toward Understanding, Valuing, and Informing the Therapeutic Role of Foster Parents

Foster parents have the potential to effect lasting therapeutic change through their role with the children they temporarily foster. Therapists working with foster parents can understand, support, and inform foster parents in their role based on a commonality that exists between the roles of therapists and foster parents. Similarities at different stages of both the therapeutic and foster parenting relationships are addressed, as well as the use of these relationships in a therapeutic manner. Advantages (for foster parents, foster children, therapists, and the foster care system) of articulating the foster parenting relationship through the lens of the therapeutic relationship are also discussed. Future research into the experience of foster parents in their role will be essential in creating an effective and sustainable system of care for vulnerable children.

Role of potassium orbitals in the metallic behavior of K3picene

Detailed electronic structure calculations of picene clusters doped by potassium modeling the crystalline K3picene structure show that while two electrons are completely transferred from potassium atoms to the lowest-energy unoccupied molecular orbital of pristine picene, the third one remains closely attached to both material components. Multiconfigurational analysis is necessary to show that many structures of almost degenerate total energies compete to define the cluster ground state. Our results prove that the 4s orbital of potassium should be included in any interaction model describing the material. We propose a quarter-filled two-orbital model as the most simple model capable of describing the electronic structure of K-intercalated picene. Precise solutions obtained by a development of the Lanczos method show low-energy electronic excitations involving orbitals located at different positions. Consequently, metallic transport is possible in spite of the clear dominance of interaction over hopping.