Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex1

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O2-evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two protein-translocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.

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.

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.

Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers.

A soluble form of Alzheimer disease amyloid beta-protein (sA beta) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA beta (1-40)-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA beta (1-40)-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA beta (1-40)-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA beta (1-40)-apoJ complex at the cerebral vascular endothelium and choroid epithelium.

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.

Transport of cytoskeletal elements in the squid giant axon.

In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.

Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.

Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons.

Transmission and Transport of Energy in the Western U.S. and Canada: A Law and Policy Road Map

This collection of short essays arose from the inaugural meeting of the Idaho Symposium on Energy in the West, which was held in November, 2014. The topic for this first Symposium was Transmission and Transport of Energy in the Western U.S. and Canada: A Law and Policy Road Map. The essays in this collection provide a notable introduction to the major energy issues facing the West today. Topics include: building a resilient legal architecture for western energy production; natural gas flaring; transmission planning for wind energy; utilities and rooftop solar; special considerations for western states and the Clean Power Plan; the Clean Power Plan's implications for the western grid; siting renewable energy on public lands; and implications of utility reform in New York and Hawaii for the Northwest.

Spin depolarization in the transport of holes across GaxMn1−xAs∕GayAl1−yAs∕p-GaAs

We study the spin polarization of tunneling holes injected from ferromagnetic GaMnAs into a p-doped semiconductor through a tunneling barrier. We find that spin-orbit interaction in the barrier and in the drain limits severely spin injection. Spin depolarization is stronger when the magnetization is parallel to the current than when it is perpendicular to it.

Fate and Transport of Naproxen in a Sandy Aquifer Material: Saturated Column Studies and Model Evaluation

Naproxen-C14H14O3 is a nonsteroidal anti-inflammatory drug which has been found at detectable concentrations in wastewater, surface water, and groundwater. Naproxen is relatively hydrophilic and is in anionic form at pH between 6 and 8. In this study, column experiments were performed using an unconsolidated aquifer material from an area near Barcelona (Spain) to assess transport and reaction mechanisms of Naproxen in the aquifer matrix under different pore water fluxes. Results were evaluated using HYDRUS-1D, which was used to estimate transport parameters. Batch sorption isotherms for Naproxen conformed with the linear model with a sorption coefficient of 0.42 (cm3 g−1), suggesting a low sorption affinity. Naproxen breakthrough curves (BTCs) measured in soil columns under steady-state, saturated water flow conditions displayed similar behavior, with no apparent hysteresis in sorption or dependence of retardation (R, 3.85-4.24) on pore water velocities. Soil sorption did not show any significant decrease for increasing flow rates, as observed from Naproxen recovery in the effluent. Sorption parameters estimated by the model suggest that Naproxen has a low sorption affinity to aquifer matrix. Most sorption of Naproxen occurred on the instantaneous sorption sites, with the kinetic sorption sites representing only about 10 to 40% of total sorption.

THE TRANSPORT OF HEAT BY FLOWING GROUNDWATER IN A DISCRETE FRACTURE

In typical theoretical or experimental studies of heat migration in discrete fractures, conduction and thermal dispersion are commonly neglected from the fracture heat transport equation, assuming heat conduction into the matrix is predominant. In this study analytical and numerical models are used to investigate the significance of conduction and thermal dispersion in the plane of the fracture for a point and line sources geometries. The analytical models account for advective, conductive and dispersive heat transport in both the longitudinal and transverse directions in the fracture. The heat transport in the fracture is coupled with a matrix equation in which heat is conducted in the direction perpendicular to the fracture. In the numerical model, the governing heat transport processes are the same as the analytical models; however, the matrix conduction is considered in both longitudinal and transverse directions. Firstly, we demonstrate that longitudinal conduction and dispersion are critical processes that affect heat transport in fractured rock environments, especially for small apertures (eg. 100 μm or less), high flow rate conditions (eg. velocity greater than 50 m/day) and early time (eg. less than 10 days). Secondly, transverse thermal dispersion in the fracture plane is also observed to be an important transport process leading to retardation of the migrating heat front particularly at late time (eg. after 40 days of hot water injection). Solutions which neglect dispersion in the transverse direction underestimate the locations of heat fronts at late time. Finally, this study also suggests that the geometry of the heat sources has significant effects on the heat transport in the system. For example, the effects of dispersion in the fracture are observed to decrease when the width of the heat source expands.