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Atg22 recycles amino acids to link the degradative and recycling functions of autophagy.

In response to stress conditions (such as nutrient limitation or accumulation of damaged organelles) and certain pathological situations, eukaryotic cells use autophagy as a survival mechanism. During nutrient stress the main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol. This study elucidates a molecular mechanism for linking the degradative and recycling roles of autophagy. We show that in contrast to published studies, Atg22 is not directly required for the breakdown of autophagic bodies within the lysosome/vacuole. Instead, we demonstrate that Atg22, Avt3, and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagic degradation. The release of autophagic amino acids allows the maintenance of protein synthesis and viability during nitrogen starvation. We propose a "recycling" model that includes the efflux of macromolecules from the lysosome/vacuole as the final step of autophagy.

Amino Acid Transport Systems↗

The antibody to GD3 ganglioside, R24, is rapidly endocytosed and recycled to the plasma membrane via the endocytic recycling compartment. Inhibitory effect of brefeldin A and monensin.

Gangliosides are sialic acid-containing glycosphingolipids present on mammalian plasma membranes, where they participate in cell-surface events such as modulation of growth factor receptors and cell-to-cell and cell-to-matrix interactions. Antibodies to gangliosides have been associated with a wide range of clinically identifiable acute and chronic neuropathy syndromes. In addition, antibodies to tumor-associated gangliosides are being used as therapeutic agents. Their binding to and release from cell membranes and intracellular destinations have not so far been extensively examined. In this study, we characterized in both GD3 ganglioside-expressing Chinese hamster ovary (CHO)-K1 and SK-Mel 28 melanoma cells the intracellular trafficking and subcellular localization of the mouse monoclonal antibody to GD3, R24. By biochemical techniques and detailed confocal microscopic analysis, we demonstrate that the GD3-R24 antibody complex is rapidly and specifically internalized by a dynamin 2-independent pathway and then accumulates in the endocytic recycling compartment. In addition, we show that the R24 antibody exits the recycling compartment en route to the plasma membrane by a dynamin 2-dependent pathway sensitive to brefeldin A and monensin. Taken together, our results indicate that the GD3-R24 complex is endocytosed in GD3-expressing cells, accumulates in the recycling endosome, and is transported back to the plasma membrane via a route that involves clathrin-coated vesicles.

Animals↗

Differences in the osmotic fragility of recycling and reserve synaptic vesicles from the cholinergic electromotor nerve terminals of Torpedo and their possible significance for vesicle recycling.

In this study we demonstrate differences in the osmotic fragility of two metabolically and physically heterogeneous synaptic vesicle populations from stimulated electromotor nerve terminals. When synaptic vesicles isolated on sucrose density gradients are submitted to solutions of decreasing osmolarity 50% of VP2-type vesicles lysed at (mean + S.E. (number of experiments] 332 +/- 14 (4) mosM and 50% of VP1-type vesicles lysed at 573 +/- 8 (3) mosM. These results indicate that recycling vesicles are more resistant to hypo-osmotic lysis and they are consistent with our earlier conclusion that changes in water content on recycling are secondary to changes in the content of the osmotically active small-molecular-mass constituents acetylcholine and ATP.

Acetylcholine↗

Recycled vertical flow constructed wetland (RVFCW)--a novel method of recycling greywater for irrigation in small communities and households.

The use of greywater for irrigation is becoming increasingly common. However, raw greywater is often contaminated and can cause environmental harm and pose health risks. Nevertheless, it is often used without any significant pretreatment, a practice mistakenly considered safe. The aim of this study was to develop an economically sound, low-tech and easily maintainable treatment system that would allow safe and sustainable use of greywater for landscape irrigation in small communities and households. The system is based on a combination of vertical flow constructed wetland with water recycling and trickling filter, and is termed recycled vertical flow constructed wetland (RVFCW). The RVFCW's properties, removal efficiency, hydraulic parameters and feasibility were studied, as well as the environmental effects of the treated greywater, as reflected by soil and plant parameters over time. The RVFCW was efficient at removing virtually all of the suspended solids and biological oxygen demand, and about 80% of the chemical oxygen demand after 8h. Fecal coliforms dropped by three to four orders of magnitude from their initial concentration after 8h, but this was not always enough to meet current regulations for unlimited irrigation. The treated greywater had no significant negative impact on plants or soil during the study period. The feasibility analysis indicated a return over investment after approximately three years. We concluded that the RVFCW is a sustainable and promising treatment system for greywater use that can be run and maintained by unskilled operators.

Agriculture↗

Super blue box recycling (SUBBOR) enhanced two-stage anaerobic digestion process for recycling municipal solid waste: laboratory pilot studies.

The super blue box recycling (SUBBOR) process is an enhanced, multi-stage anaerobic digestion process for mixed municipal solid waste (MSW) and other biomass feedstock materials. The technology centers on enhanced high solids, thermophilic digestion after steam-pressure disruption of the ligno-cellulosic fiber components that are recalcitrant to conventional anaerobic digestion. Mixed MSW, rich in organic components but also containing inorganic materials, such as glass, aluminum and steel, as well as non-digestible plastic materials, has been laboratory pilot tested with a fully integrated process train designed to treat and recycle all of the MSW components. Methane yields from the MSW were 0.36 m3 CH4/kg volatile solids (VS) representing a 40% increase over the yield obtained from conventional single stage digestion. The secondary digestion step after steam pressure disruption also provided a 40% improvement in total solids and VS reduction. The residual organic fraction following two-stage digestion was fine in texture and was recovered as a clean peat fraction with reduced contents of heavy metal and other fugitive non-digested contaminants. Mass and energy balance determinations indicated a high degree of MSW diversion from landfill disposal (>80%) was achievable by the SUBBOR process as well as substantial net electrical and thermal energy production. Continuous long-term trials of the SUBBOR process at 25,000 tonnes/year are underway.

Anaerobiosis↗

Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in escherichia coli carrying temperature-sensitive ribosome recycling factor.

We have isolated a protein, mature RRFHCP, from chloroplasts of spinach (Spinacia oleracea L.) that shows 46% sequence identity and 66% sequence homology with ribosome recycling factor (RRF) of Escherichia coli. RRF recycles ribosomes through disassembly of the posttermination complex. From the cDNA analysis and from the amino-terminal sequencing of the isolated protein, the mature RRFHCP was deduced to have a Mr of 21,838 with 193 aa. It lacks the 78-aa chloroplast targeting sequence encoded by the RRFHCP cDNA sequence. The RRFHCP synthesized in vitro was imported into isolated chloroplasts with simultaneous conversion to the mature RRFHCP. Transcription of the gene coding for RRFHCP was not dependent on light, yet it was limited mostly to photosynthetic tissues in which only one transcript size was detected. Mature RRFHCP exerted a bactericidal effect on E. coli carrying temperature-sensitive RRF at the permissive temperature whereas wild-type E. coli was not affected.

Amino Acid Sequence↗

Phorbol esters target the activity-dependent recycling pool and spare spontaneous vesicle recycling.

Using electrophysiology and styryl dye imaging, we studied the effect of phorbol 12-myristate 13-acetate (PMA) on activity-dependent and spontaneous vesicle recycling. In electrophysiological experiments, we found that the PMA effect depended on the maturational state of the synapses. Spontaneous neurotransmitter release from nascent synapses without a functional readily releasable pool (RRP) was unresponsive to PMA application. In contrast, mature synapses responded robustly to PMA application, consistent with previous studies. Using styryl dye imaging, we found that there was a PMA-dependent increase in the size of the RRP when PMA was present before, during, or after activity-dependent dye uptake, suggesting that this effect involves an increase in the population of the RRP by vesicles recruited from the reserve pool. Additionally, we found that when PMA was present during spontaneous dye uptake, there was an increase in dye labeling, and these additional dye-loaded vesicles showed rapid destaining in response to strong stimulation and were also releasable by hypertonic sucrose. In contrast, these observations were not reproducible when PMA treatment was performed after spontaneous dye uptake and extracellular dye washout. Together, these findings suggest that the increased spontaneous neurotransmission in the presence of PMA was attributable to release of vesicles from the RRP rather than an effect of PMA on the spontaneously recycling pool. Thus, the phorbol esters selectively regulate the activity-dependent pool of vesicles, indicating that priming mechanisms that prepare vesicles for fusion, which are targeted by phorbol esters, are different for the spontaneous and evoked forms of fusion.

Animals↗

Internalization and recycling of transferrin and its receptor. Effect of trifluoperazine on recycling in human erythroleukemic cells.

When human erythroleukemic (K562) cells were incubated with 25 microM trifluoperazine (TFP), a drug that inhibits both calmodulin-dependent and calcium-activated phospholipid-dependent kinases, the number of transferrin receptors detected on the cell surface was reduced to approximately half with no change in the affinity of the remaining surface receptors. Removal of the TFP from the incubation medium reversed the loss of surface receptors and they returned to the cell surface in an apparently synchronous manner. As a result, the number of receptors detected on the cell surface exceeded the original level but later returned to normal. Measurements of the total number of receptors available to transferrin in TFP-treated cells suggested that the lost receptors were not participating in the internalization and recycling pathway but instead were probably trapped at an intracellular location. However, those receptors that remained on the cell surface continued to internalize transferrin and to recycle apotransferrin to the cell surface albeit more slowly than in cells that had not been treated with TFP. Using transferrin that had been labeled with iron-59, it was found that although iron uptake was reduced in line with the diminished number of surface receptors, iron still accumulated within TFP-treated cells, suggesting that in the presence of the drug, transferrin-transferrin receptor complexes continued to migrate through an intracellular compartment that contained a low pH.

Cells, Cultured↗

Specific interaction between the ribosome recycling factor and the elongation factor G from Mycobacterium tuberculosis mediates peptidyl-tRNA release and ribosome recycling in Escherichia coli.

Once the translating ribosomes reach a termination codon, the nascent polypeptide chain is released in a factor-dependent manner. However, the P-site-bound deacylated tRNA and the ribosomes themselves remain bound to the mRNA (post-termination complex). The ribosome recycling factor (RRF) plays a vital role in dissociating this complex. Here we show that the Mycobacterium tuberculosis RRF (MtuRRF) fails to rescue Escherichia coli LJ14, a strain temperature-sensitive for RRF (frr(ts)). More interestingly, co-expression of M.tuberculosis elongation factor G (MtuEFG) with MtuRRF rescues the frr(ts) strain of E.coli. The simultaneous expression of MtuEFG is also needed to cause an enhanced release of peptidyl-tRNAs in E.coli by MtuRRF. These observations provide the first genetic evidence for a functional interaction between RRF and EFG. Both the in vivo and in vitro analyses suggest that RRF does not distinguish between the translating and terminating ribosomes for their dissociation from mRNA. In addition, complementation of E.coli PEM100 (fusA(ts)) with MtuEFG suggests that the mechanism of RRF function is independent of the translocation activity of EFG.

Amino Acid Sequence↗

Nutrient recycling systems of Biosphere 2. Litterfall, decomposition, and wastewater recycling: results from the 1991-1993 closure experiment.

Important aspects of nutrient recycling in Biosphere 2 were plant litterfall/leaf decomposition in the terrestrial biomes and sewage and wastewater processing by a constructed wetland system. During the second year of closure, annual litterfall ranged from 1317 +/- 283 g/m2 in the lowland rainforest to 141 +/- 58 g/m2 in the desert sand dune area. Litterfall patterns followed different seasonal regimes in the biomes. Leaf decomposition averaged 90%/year in Biosphere 2, with the smallest leaf loss in the desert biome. For maintenance of soil fertility in the agricultural area, inedible crop material and domestic animal manure was composted, and human waste products and wastewater from people, labs, and domestic animals was processed in a 41-m2 constructed wetland. The wetland produced 1200 kg of fodder and remaining nutrients were returned to the soils via irrigation water.

Biomass↗

[A long-term field trial on fertilization and on use of recycled nutrients in farming systems III. Recycling rate of P and N through a composting cycle and recoveries of P and N in organic manure].

The results from a nine years experiment indicated that the average loss rate of N and P contained in harvest products through a feeding-composting cycle was about 30%. Under high yield conditions, the amount of recycled N and P from 60% of harvested products and through a composting cycle in a farming system were about 55-70 kg.hm-2 and 9-11 kg.hm-2, respectively, equivalent to 37%-47% of N and 36%-44% of P from fertilizers applied every year to the system.

Agriculture↗

Direct demonstration of rapid insulin-like growth factor II Receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process.

The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl-125I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl-125I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The low density microsomes were previously shown to contain intracellular membranes (Oka, Y., and Czech, M.P. (1984) J. Biol. Chem. 259, 8125-8133). The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II, measured by the binding of anti-IGF-II receptor antibody to cells, remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time, as evidenced by a large increase in the photolabeling of intracellular membrane IGF-II receptors when cells are incubated at 37 degrees C with insulin and 4-azidobenzoyl-125I-IGF-II prior to photoactivation; and 3) increases the rate of cellular 125I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody. The results indicate that the action of insulin to elevate the steady state number of cell surface IGF-II receptors leads to an increased internalization flux of IGF-II-bound receptors, mediating increased IGF-II uptake and degradation.

Adipose Tissue↗

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis.

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.

Adenosine Triphosphate↗