The small GTPase Arf1 regulates ATP synthesis and mitochondria homeostasis by modulating fatty acid metabolism

Lipid mobilization through fatty acid β-oxidation is a central process essential for energy 36 production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where β-oxidation products enter mitochondria and fuel the TCA cycle. Little is known about the physical and metabolic cooperation between these organelles. We found that expression of fatty acid transporters and of the rate-limiting enzyme involved in β-oxidation are decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. As a consequence, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although β-oxidation occurs mainly in mitochondria in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact-sites

Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts

The ArfGAP2/3 Glo3 and ergosterol collaborate in transport of a subset of cargoes

Proteins reach the plasma membrane through the secretory pathway in which the trans Golgi network (TGN) acts as a sorting station. Transport from the TGN to the plasma membrane is maintained by a number of different pathways that act either directly or via the endosomal system. Here we show that a subset of cargoes depends on the ArfGAP2/3 Glo3 and ergosterol to maintain their proper localization at the plasma membrane. While interfering with neither ArfGAP2/3 activity nor ergosterol biosynthesis individually significantly altered plasma membrane localization of the tryptophan transporter Tat2, the general amino acid permease Gap1 and the v-SNARE Snc1, in a ∆glo3 ∆erg3 strain those proteins accumulated in internal endosomal structures. Export from the TGN to the plasma membrane and recycling from early endosomes appeared unaffected as the chitin synthase Chs3 that travels along these routes was localized normally. Our data indicate that a subset of proteins can reach the plasma membrane efficiently but after endocytosis becomes trapped in endosomal structures. Our study supports a role for ArfGAP2/3 in recycling from endosomes and in transport to the vacuole/lysosome

Head structure of bacteriophages T2 and T4

The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods. The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis. Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*

Dominant pro-vasopressin mutants that cause diabetes insipidus form disulfide-linked fibrillar aggregates in the endoplasmic reticulum

Autosomal dominant neurohypophyseal diabetes insipidus results from mutations in the precursor protein of the antidiuretic hormone arginine vasopressin. Mutant prohormone is retained in the endoplasmic reticulum of vasopressinergic neurons and causes their progressive degeneration by an unknown mechanism. Here, we show that several dominant pro-vasopressin mutants form disulfide-linked homo-oligomers and develop large aggregations visible by immunofluorescence and immunogold electron microscopy, both in a fibroblast and a neuronal cell line. Double-labeling showed the pro-vasopressin aggregates to colocalize with the chaperone calreticulin, indicating that they originated from the endoplasmic reticulum. The aggregates revealed a remarkable fibrillar substructure. Bacterially expressed and purified mutant pro-vasopressin spontaneously formed fibrils under oxidizing conditions. Mutagenesis experiments showed that the presence of cysteines, but no specific single cysteine, is essential for disulfide oligomerization and aggregation in vivo. Our findings assign autosomal dominant diabetes insipidus to the group of neurodegenerative diseases associated with the formation of fibrillar protein aggregates

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

In endocrine cells, prohormones and granins are segregated in the trans-Golgi network from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner upon specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures which resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of chromogranin A is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP or a short epitope tag was expressed in COS-1 fibroblast cells and analyzed by fluorescence and electron microscopy and pulse-chase labeling. Full-length chromogranin A as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that colocalized with coexpressed secretogranin II. These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway

A methionine synthase homolog is associated with secretory vesicles in tobacco pollen tubes

Seven isoforms of 85 kDa polypeptides (p85) were identified as methionine synthase (MetE) homologs by partial aminoacid sequencing in tobacco pollen tube extracts. Immunocytochemistry data showed a localization of the antigen on the surface of tip-focussed post-Golgi secretory vesicles (SVs), that appear to be partially associated with microtubules (Mts). The chemical dissection of pollen tube high speed supernatant (HSS) showed that two distinct pools of MetE are present in pollen tubes, one being the more acidic isoforms sedimenting at 15S and the remaining at 4S after zonal centrifugation through a sucrose density gradient. The identification of the MetE within the pollen tube and its possible participation as methyl donor in a wide range of metabolic reactions, makes it a good subject for studies on pollen tube growth regulation