Using Two-Dimensional Distributed Feedback for Synchronization of Radiation from Two Parallel-Sheet Electron Beams in a Free-Electron Maser.

A spatially extended planar 75 GHz free-electron maser with a hybrid two-mirror resonator consisting of two-dimensional upstream and traditional one-dimensional downstream Bragg reflectors and driven by two parallel-sheet electron beams 0.8 MeV/1 kA has been elaborated. For the highly oversized interaction space (cross section 45×2.5 vacuum wavelengths), the two-dimensional distributed feedback allowed realization of stable narrow-band generation that includes synchronization of emission from both electron beams. As a result, spatially coherent radiation with the output power of 30-50 MW and a pulse duration of ∼100 ns was obtained in each channel.

Development of the 174 GHz collective Thomson scattering diagnostics at Wendelstein 7-X.

In this paper, we present the design and commissioning results of the upgraded collective Thomson scattering diagnostic at the Wendelstein 7-X stellarator. The diagnostic has a new radiometer designed to operate between the second and third harmonics of the electron cyclotron emission from the plasma at 171-177 GHz, where the emission background has a minimum and is of order 10-100 eV. It allows us to receive the scattered electromagnetic field with a significantly improved signal-to-noise ratio and extends the set of possible scattering geometries compared to the case of the original instrument operated at 140 GHz. The elements of the diagnostic are a narrowband notch filter and a frequency stabilized probing gyrotron that will allow measuring scattered radiation spectra very close to the probing frequency. Here, we characterize the microwave components applied to the radiometer and demonstrate the performance of the complete system that was achieved during the latest experimental campaign, OP2.1.

Experimental and theoretical studies of a coaxial free-electron maser based on two-dimensional distributed feedback.

The first operation of a coaxial free-electron maser (FEM) based on two-dimensional (2D) distributed feedback has been recently observed. Analytical and numerical modeling, as well as measurements, of microwave radiation generated by a FEM with a cavity defined by coaxial structures with a 2D periodic perturbation on the inner surfaces of the outer conductor were carried out. The two-mirror cavity was formed with two 2D periodic structures separated by a central smooth section of coaxial waveguide. The FEM was driven by a large diameter (7 cm), high-current (500 A), annular electron beam with electron energy of 475 keV. Studies of the FEM operation have been conducted. It has been demonstrated that by tuning the amplitude of the undulator or guide magnetic field, modes associated with the different band gaps of the 2D structures were excited. The Ka-band FEM generated 15 MW of radiation with a 6% conversion efficiency, in good agreement with theory.

A genomic screen identifies AUT8 as a novel gene essential for autophagy in the yeast Saccharomyces cerevisiae.

Autophagy is a starvation-induced transport pathway delivering parts of the cytosol into the lysosome (vacuole) for degradation. Autophagy significantly differs from other transport pathways by using double membrane layered transport intermediates. Based on the identification of autophagy genes in Saccharomyces cerevisiae, which served as a pacemaker for higher cells, our mechanistic knowledge of autophagy notably increased over the past few years. We here identify AUT8 as a novel gene essential for autophagy by screening a collection of approximately 5000 yeast deletion strains, each containing a defined deletion in an individual gene. This collection is a result of the world-wide Saccharomyces deletion project and covers the non-essential genes of the whole yeast genome. Homozygous aut8 Delta cells are impaired in maturation of proaminopeptidase I, and they fail to undergo the cell differentiation process of sporulation. The essential function of AUT8 for autophagy is further demonstrated by the lack of accumulation of autophagic vesicles in the vacuoles of aut8 Delta cells starved of nitrogen in the presence of the proteinase B inhibitor phenylmethylsulfonyl fluoride.

Catabolite inactivation of fructose-1,6-bisphosphatase in yeast is mediated by the proteasome.

Fructose-1,6-bisphosphatase, a key enzyme in gluconeogenesis, undergoes catabolite inactivation when glucose is added to gluconeogenetically active cells of the yeast Saccharomyces cerevisiae. Phosphorylation of the enzyme is followed by rapid degradation. To elucidate the cellular proteolytic system involved in catabolite-triggered degradation of fructose-1,6-bisphosphatase this event was followed in different protease-deficient yeast mutants. In a mutant defective in the proteolytic function of the vacuole the degradation rate of the enzyme is not diminished. In contrast mutants defective in the proteolytic activity of the proteasome exhibit a strongly reduced glucose-induced degradation of fructose-1,6-bisphosphatase as compared to their isogenic wild-type counterparts. Our studies suggest that catabolite inactivation of fructose-1,6-bisphosphatase occurs in the cytosol, the degradation event being mediated by the proteasome. An explanation is presented which tries to resolve the formerly conflicting results, which suggested glucose-triggered uptake of fructose-1,6-bisphosphatase into the vacuole followed by vacuolar proteolysis.

Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p.

We here report the identification of AUT10 as a novel gene required for both the cytoplasm to vacuole targeting of proaminopeptidase I and starvation-induced autophagy. aut10Delta cells are impaired in maturation of proaminopeptidase I under starvation and non-starvation conditions. A lack of Aut10p causes a defect in autophagy prior to vacuolar uptake of autophagosomes. Homozygous aut10Delta diploids do not sporulate. Vacuolar acidification indicated by accumulation of quinacrine is normal in aut10Delta cells and mature vacuolar proteinases are present. A biologically active Ha-tagged Aut10p, chromosomally expressed from its endogenous promoter, localizes in indirect immunofluorescence microscopy in the cytosol and on granulated structures, which appear clustered around the vacuolar membrane. This localization differs from known autophagy proteins.

Isolation of autophagocytosis mutants of Saccharomyces cerevisiae.

Protein degradation in the vacuole (lysosome) is an important event in cellular regulation. In yeast, as in mammalian cells, a major route of protein uptake for degradation into the vacuole (lysosome) has been found to be autophagocytosis. The discovery of this process in yeast enables the elucidation of its mechanisms via genetic and molecular biological investigations. Here we report the isolation of yeast mutants defective in autophagocytosis (aut mutants), using a rapid colony screening procedure.

Structure and function of the yeast vacuole and its role in autophagy.

The vacuole of the yeast Saccharomyces cerevisiae plays an important role in pH- and ion-homeostasis, and is used as a storage compartment for ions. Another important function of the vacuole, especially during nutrient limitation, is the bulk degradation of proteins and even whole organelles. To carry these proteins into the vacuolar lumen, sophisticated transport pathways have evolved. In this review, starvation-induced autophagy and its relationship to the specific cytoplasm to vacuole targeting (cvt-) pathway of proaminopeptidase I is discussed. A further topic is the specific vacuolar uptake and degradation of peroxisomes in Pichia pastoris cells via micro- and macroautophagy.

Molecular dynamics simulation of structural and dynamic properties of selenium structures with different degrees of amorphization.

We investigated Se structures of different degrees of disorder ranging from a 5% up to a 95% degree of amorphization. Starting from a trigonal crystalline structure we applied different strategies to introduce disorder into the Se configurations by irradiating atoms from their crystalline equilibrium positions. According to the symmetry of the trigonal phase, we introduced three types of disorder, i.e. the first type where only atoms forming layers of complete helical chains are shifted from their original positions (the thickness of these layers is chosen to represent the chosen degree of amorphicity), the second type where only atoms in planes-of respective thicknesses-lying perpendicular to the chains are displaced and the third type where only randomly chosen atoms are shifted from their crystalline equilibrium positions. After a thermal treatment of these disordered starting configurations, we calculated structural and dynamic properties (i.e. pair-correlation function and vibrational spectrum) and compared the results to both the original crystalline data and results obtained from corresponding glass structures.

Piecemeal microautophagy of the nucleus requires the core macroautophagy genes.

Autophagy is a diverse family of processes that transport cytoplasm and organelles into the lysosome/vacuole lumen for degradation. During macroautophagy cargo is packaged in autophagosomes that fuse with the lysosome/vacuole. During microautophagy cargo is directly engulfed by the lysosome/vacuole membrane. Piecemeal microautophagy of the nucleus (PMN) occurs in Saccharomyces cerevisiae at nucleus-vacuole (NV) junctions and results in the pinching-off and release into the vacuole of nonessential portions of the nucleus. Previous studies concluded macroautophagy ATG genes are not absolutely required for PMN. Here we report using two biochemical assays that PMN is efficiently inhibited in atg mutant cells: PMN blebs are produced, but vesicles are rarely released into the vacuole lumen. Electron microscopy of arrested PMN structures in atg7, atg8, and atg9 mutant cells suggests that NV-junction-associated micronuclei may normally be released from the nucleus before their complete enclosure by the vacuole membrane. In this regard PMN is similar to the microautophagy of peroxisomes (micropexophagy), where the side of the peroxisome opposite the engulfing vacuole is capped by a structure called the "micropexophagy-specific membrane apparatus" (MIPA). The MIPA contains Atg proteins and facilitates terminal enclosure and fusion steps. PMN does not require the complete vacuole homotypic fusion genes. We conclude that a spectrum of ATG genes is required for the terminal vacuole enclosure and fusion stages of PMN.

The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway mutants.

A P-element line ( P0997) of Drosophila melanogaster in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut ( ct) modifiers. The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy. The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants. These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway.

AUT3, a serine/threonine kinase gene, is essential for autophagocytosis in Saccharomyces cerevisiae.

Autophagocytosis is a starvation-induced process, carrying proteins destined for degradation to the lysosome. In the yeast Saccharomyces cerevisiae, the autophagic process is visualized by the appearance of autophagic vesicles in the vacuoles of proteinase yscB-deficient strains during starvation. aut3-1 mutant cells which exhibit a block in the autophagic process have been isolated previously. By using the drastically reduced sporulation frequency of homozygous aut3-1 diploid cells, the AUT3 gene was cloned by complementation. The Aut3 protein consists of 897 amino acids. The amino-terminal part of the protein shows significant homologies to serine/threonine kinases. aut3 null mutant cells are fully viable on rich media but show a reduced survival rate upon starvation. They are unable to accumulate autophagic vesicles in the vacuole during starvation. Starvation-induced vacuolar protein breakdown is almost completely impaired in aut3-deficient cells. Vacuolar morphology and acidification are not influenced in aut3-deficient cells. Also, secretion of invertase, endocytic uptake of Lucifer Yellow, and vacuolar protein sorting appear wild type like in aut3-deficient cells, suggesting autophagocytosis as a novel route for the transport of proteins from the cytosol to the vacuole. By using a fusion of Aut3p with green-fluorescent protein, Aut3p was localized to the cytosol.

Catabolite inactivation of fructose-1,6-bisphosphatase of Saccharomyces cerevisiae. Degradation occurs via the ubiquitin pathway.

Catabolite inactivation of fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis, is due to phosphorylation and subsequent degradation in the yeast Saccharomyces cerevisiae. The degradation process of the enzyme had been shown to depend on the action of the proteasome. Here we report that components of the ubiquitin pathway target FBPase to proteolysis. Upon glucose addition to yeast cells cultured on nonfermentable carbon sources FBPase is ubiquitinated in vivo. A multiubiquitin chain containing isopeptide linkages at Lys48 of ubiquitin is attached to FBPase. Formation of a multiubiquitin chain is a prerequisite for the degradation of FBPase. Catabolite degradation of FBPase is dependent on the ubiquitin-conjugating enzymes Ubc1, Ubc4, and Ubc5. The 26 S proteasome is involved in the degradation process.

Tracing intracellular proteolytic pathways. Proteolysis of fatty acid synthase and other cytoplasmic proteins in the yeast Saccharomyces cerevisiae.

Yeast fatty acid synthase consists of two independent polypeptide strains, alpha and beta. The functional multienzyme complex, composed of six alpha- and six beta-subunits, is rather stable against proteolysis in vivo. Mutations in one of the subunits or deletion of one subunit lead to degradation of the nonmutated remaining fatty acid synthase protein. We show that the unassembled alpha-subunit of this enzyme is short-lived, and degradation depends on the presence of active cytoplasmic proteinase yscE, the yeast proteasome. The unassembled beta-subunit is degraded by a nonvacuolar proteolytic system under vegetative growth conditions. However, starvation of a vacuolar proteinase mutant strain, which lacks the alpha-subunit of fatty acid synthase, leads to appearance of the unassembled beta-subunit is isolated vacuoles. This indicates that the major vacuolar peptidases proteinase yscA and yscB are at least partly involved in degradation of the beta-subunit of fatty acid synthase. In a proteinase yscA and yscB double mutant strain wild type for fatty acid synthase both subunits of fatty acid synthase, alpha and beta, are detectable in vacuoles. In addition, under the same starvation conditions other cytoplasmic proteins are found in the vacuole of a proteinase yscA and yscB double mutant strain. The experiments in conjunction with the previous finding of the appearance of vesicles in vacuoles of starved cells (Simeon, A., van der Klei, I.J., Veenhuis, M., and Wolf, D. H. (1992) FEBS Lett. 301, 231-235) indicate that transport of these tested cytoplasmic proteins into the vacuole is an unselective bulk process induced by nutritional stress.

Genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein targeting pathway.

We have explored the phenotypic and genetic overlap between autophagocytosis and cytoplasm to vacuole targeting in the yeast Saccharomyces cerevisiae. Complementation analysis was performed with mutants in each of these groups (aut and cvt, respectively), and three complementation groups were found to overlap. Also, most of the unique aut mutants accumulated precursor aminopeptidase I in the cytoplasm, while maintaining wild type kinetics and maturation of proteins targeted to the vacuole via the secretory pathway. The majority of the non-overlapping cvt mutants were found to be at least partially defective in autophagy. Some mutants in each group, however, appear to be only marginally affected in the other phenotype, implying that these pathways only partially overlap. We propose that import of aminopeptidase I into the vacuole shares a number of components required for bulk autophagocytosis, but is made specific, saturable, and constitutive by the presence of a receptor or other interacting protein(s).

AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae.

Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole. In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB. The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized. AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa. Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole. aut1 null mutant strains are completely viable but show decreased survival rates during starvation. Homozygous delta aut1 diploid cells fail to sporulate. The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells. Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting. This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole.

Aut2p and Aut7p, two novel microtubule-associated proteins are essential for delivery of autophagic vesicles to the vacuole.

AUT2 and AUT7, two novel genes essential for autophagocytosis in the yeast Saccharomyces cerevisiae were isolated. AUT7 was identified as a low copy suppressor of autophagic defects in aut2-1 cells. Aut7p is a homologue of the rat microtubule-associated protein (MAP) light chain 3 (LC3). Aut2p and Aut7p interact physically. Aut7p is attached to microtubules via Aut2p, which interacts with tubulins Tub1p and Tub2p. aut2- and aut7-deleted cells are unable to deliver autophagic vesicles and the precursor of aminopeptidase I to the vacuole. Double membrane-layered autophagosome-like vesicles accumulate in the cytoplasm of these cells. Our findings suggest that microtubules and an attached protein complex of Aut2p and Aut7p are involved in the delivery of autophagic vesicles to the vacuole.

Ubc8p functions in catabolite degradation of fructose-1, 6-bisphosphatase in yeast.

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is synthesized when cells of the yeast Saccharomyces cerevisiae are grown on a non-fermentable carbon source. After shifting the cells to glucose-containing medium, in a process called catabolite degradation, FBPase is selectively and rapidly broken down. We have isolated gid mutants, which are defective in this glucose-induced degradation process. When complementing the defect in catabolite degradation of FBPase in gid3-1 mutant cells with a yeast genomic library, we identified the GID3 gene and found it to be identical to UBC8 encoding the ubiquitin-conjugating enzyme Ubc8p. The in vivo function of Ubc8p (Gid3p) has remained a mystery so far. Here we demonstrate the involvement of Ubc8p in the glucose-induced ubiquitylation of FBPase as a prerequisite for catabolite degradation of the enzyme via the proteasome. Like FBPase, Ubc8p is found in the cytoplasmic fraction of the cell. We demonstrate cytoplasmic degradation of FBPase.

Aut5/Cvt17p, a putative lipase essential for disintegration of autophagic bodies inside the vacuole.

Selective disintegration of membrane-enclosed autophagic bodies is a feature of eukaryotic cells not studied in detail. Using a Saccharomyces cerevisiae mutant defective in autophagic-body breakdown, we identified and characterized Aut5p, a glycosylated integral membrane protein. Site-directed mutagenesis demonstrated the relevance of its putative lipase active-site motif for autophagic-body breakdown. aut5Delta cells show reduced protein turnover during starvation and are defective in maturation of proaminopeptidase I. Most recently, by means of the latter phenotype, Aut5p was independently identified as Cvt17p. In this study we additionally checked for effects on vacuolar acidification and detected mature vacuolar proteases, both of which are prerequisites for autophagic-body lysis. Furthermore, biologically active hemagglutinin-tagged Aut5p (Aut5-Ha) localizes to the endoplasmic reticulum (nuclear envelope) and is targeted to the vacuolar lumen independent of autophagy. In pep4Delta cells immunogold electron microscopy located Aut5-Ha at approximately 50-nm-diameter intravacuolar vesicles. Characteristic missorting in vps class E and fab1Delta cells, which affects the multivesicular body (MVB) pathway, suggests vacuolar targeting of Aut5-Ha similar to that of the MVB pathway. In agreement with localization of Aut5-Ha at intravacuolar vesicles in pep4Delta cells and the lack of vacuolar Aut5-Ha in wild-type cells, our pulse-chase experiments clearly indicated that Aut5-Ha degradation with 50 to 70 min of half-life is dependent on vacuolar proteinase A.

The breakdown of autophagic vesicles inside the vacuole depends on Aut4p.

Autophagy is a degradative transport pathway that delivers cytosolic proteins to the lysosome (vacuole). Cytosolic proteins appear inside the vacuole enclosed in autophagic vesicles. These autophagic vesicles are broken down in the vacuole together with their cytosolic content. The breakdown of vesicular transport intermediates is a unique feature of autophagy. We here identify Aut4p, a component essential for the disintegration of autophagic vesicles, inside the vacuole of S. cerevisiae cells. Aut4p is a putative integral membrane protein with limited homologies to permeases. Chromosomal deletion of AUT4 has no obvious influence on growth, vacuolar acidification and the activities of vacuolar proteinases. Like proteinase B-deficient cells, aut4-deleted cells show a partial reduction in total protein breakdown during nitrogen starvation. A biologically active fusion protein of Aut4p and the green fluorescent protein is visualized at the vacuolar membrane and in punctate structures attached to the vacuole.