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The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and addressing to membranes through a conjugation to a lipid, the phosphatidylethanolamine. Contrarily to mammals, C. elegans possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosomal membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in C. elegans, but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy independent embryonic function. Our data question the use of the lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.

The yeast N-BAR (Bin/Amphiphysin/Rvs167) protein Rvs167 is recruited by the Rab GTPase Activating Proteins (RabGAP) Gyp5 and Gyl1 to the tip of small buds to act in exocytosis. Investigating other N-BAR proteins involved in Gyp5/Gyl1/Rvs167 complexes, we found that Rvs161, an Rvs167 paralog, is absent from the complexes formed at the tip of small buds. Immunoprecipitation and Bimolecular Fluorescence Complementation (BiFC) analysis show that both Rvs167 and Rvs161 interact in vivo with Gvp36, an N-BAR protein. Rvs167 molecules also interact independently of Rvs161 and Gvp36. Rvs167/Rvs167 and Rvs167/Gyp5 interactions predominate over other combinations at the tip of small buds, suggesting that N-BAR lattices enriched in Rvs167 molecules form at these sites. By combining BiFC with markers specific to each organelle, we analyzed systematically in living cells the locations of the BiFC signals generated by combinations of the three N-BAR proteins. We show that the BiFC signals differ according to organelle and cell site, strongly suggesting heterogeneity in the composition of N-BAR protein lattices in vivo. Our results reveal that the organization of N-BAR protein lattices in vivo is complex and are consistent with N-BAR proteins forming various types of dimers and lattices of variable composition.

Background information: During phagocytosis, neutrophils internalize pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH oxidase subunits p40phox, p47phox, p67phox and Rac2 translocate to the phagosomal membrane to participate in enzyme activation. The kinetics of this recruitment and the underlying signalling pathways are only partially understood. Anionic phospholipids, phosphatidylserine (PS) and phosphoinositides (PPI) provide important attachment site for numerous proteins, including several oxidase subunits. Results: We investigated the kinetics of p47phox and Rac2 phagosomal membrane recruitment. Both subunits are known to interact with anionic phospholipids; we therefore addressed the role of PS in this recruitment. Phagosomal accumulation of p47phox and Rac2 tagged with fluorescent proteins was analyzed by videomicroscopy. We used the C2 domain of lactadherin (lactC2) that interacts strongly and specifically with PS to monitor intracellular PS localization and to decrease PS accessibility. During phagocytosis of opsonized zymosan, p47phox and constitutively active Rac2G12V briefly translocated to the phagosomal membrane while ROS production continued for a longer period. However, in the presence of lactC2, Rac2G12V recruitment was inhibited and the kinetics of p47phox recruitment and detachment were delayed. A reduced phagosomal ROS production was also observed during the first seven minutes following the phagosome closure. Conclusions: These results suggest that p47phox and Rac2 accumulate only transiently at the phagosome at the onset of NADPH activity and detach from the phagosome before the end of ROS production. Furthermore, lactC2, by masking PS, interfered with the phagosomal recruitment of p47phox and Rac2 and disturbed NADPH oxidase activity. Thus, PS appears as a modulator of NADPH oxidase activation.

The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway mainly expressed in the liver. Rat liver contains a regulatory protein that inhibits glucokinase (GK) activity. The effect of this protein is greatly reinforced by the fructose 6-phosphate and antagonized by the fructose 1-phosphate (Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184). In hepatocytes, fructose in low concentrations is phosphorylated into fructose 1-phosphate, and therefore is able to active GK in the absence of insulin via the regulatory protein in the liver. In primary culture of rat hepatocytes, 0.2 mM fructose in the presence of 20 or 40 mM glucose stimulated the activity of the L-PK gene promoter fused with the chloramphenicol acetyltransferase reporter gene, regardless of the addition of insulin, through the glucose/insulin response element. A constitutive GK expression vector co-transfected with the L-PK/chloramphenicol acetyltransferase construct is also able to confer an insulin-independent glucose responsiveness in hepatocytes. Thus, the insulin effect on glucose-dependent activation of the L-PK promoter is, under these experimental conditions, to permit glucose phosphorylation through the stimulation of the GK synthesis. In the presence of glucose, the L-PK promoter can also be activated by a post-translational GK activation, mediated by a low concentration of fructose acting via the regulatory protein of glucokinase.

OBJECTIVES—A physiological adaptation to a sugar-rich meal is achieved by increased sugar uptake to match dietary load, resulting from a rapid transient translocation of the fructose/glucose GLUT2 transporter to the brush border membrane (BBM) of enterocytes. The aim of this study was to define the contributors and physiological mechanisms controlling intestinal sugar absorption, focusing on the action of insulin and the contribution of GLUT2-mediated transport. RESEARCH DESIGN AND METHODS—The studies were performed in the human enterocytic colon carcinoma TC7 subclone (Caco-2/TC7) cells and in vivo during hyperinsulinemic-euglycemic clamp experiments in conscious mice. Chronic high-fructose or high-fat diets were used to induce glucose intolerance and insulin resistance in mice. RESULTS AND CONCLUSIONS—In Caco-2/TC7 cells, insulin action diminished the transepithelial transfer of sugar and reduced BBM and basolateral membrane (BLM) GLUT2 levels, demonstrating that insulin can target sugar absorption by controlling the membrane localization of GLUT2 in enterocytes. Similarly, in hyperinsulinemic-euglycemic clamp experiments in sensitive mice, insulin abolished GLUT2 (i.e., the cytochalasin B-sensitive component of fructose absorption), decreased BBM GLUT2, and concomitantly increased intracellular GLUT2. Acute insulin treatment before sugar intake prevented the insertion of GLUT2 into the BBM. Insulin resistance in mice provoked a loss of GLUT2 trafficking, and GLUT2 levels remained permanently high in the BBM and low in the BLM. We propose that, in addition to its peripheral effects, insulin inhibits intestinal sugar absorption to prevent excessive blood glucose excursion after a sugar meal. This protective mechanism is lost in the insulin-resistant state induced by high-fat or high-fructose feeding.

Glucose catabolism induces the expression of the L-type pyruvate kinase (L-PK) gene through the glucose response element (GIRE). The metabolic pathway used by glucose after its phosphorylation to glucose 6-phosphate by glucokinase to induce L-PK gene expression in hepatocytes remains unknown. The sugar alcohol xylitol is metabolized to xylulose 5-phosphate, an intermediate of the nonoxidative branch of the pentose phosphate pathway. In this study, we demonstrated that xylitol at low concentration (O.5 mM) induced the expression of the L-PK/CAT construct in glucose-responsive mhAT3F hepatoma cells at the same level as 20 mM glucose, while it did not affect intracellular concentration of glucose 6-phosphate significantly. The effect of xylitol on the induction of the L-PK gene expression was noncumulative with that of glucose since 20 mM glucose plus 5 mM xylitol induced the expression of the L-PK/CAT construct similarly to 20 mM glucose alone. In hepatocytes in primary culture, 5 mM xylitol induced accumulation of the L-PK mRNA even in the absence of insulin. Furthermore, the response to xylitol as well as glucose required the presence of a functional GIRE. It can be assumed from these results that glucose induces the expression of the L-PK gene through the nonoxidative branch of the pentose phosphate pathway. The effect of xylitol at low concentration suggests that the glucose signal to the transcriptional machinery is mediated by xylulose 5-phosphate.