Atg21p is essential for macropexophagy and microautophagy in the yeast Hansenula polymorpha

ATG genes are required for autophagy-related processes that transport proteins/organelles destined for proteolytic degradation to the vacuole. Here, we describe the identification and characterisation of the Hansenula polymorpha ATG21 gene. Its gene product Hp-Atg21p, fused to eGFP, had a dual location in the cytosol and in peri-vacuolar dots. We demonstrate that Hp-Atg21p is essential for two separate modes of peroxisome degradation, namely glucose-induced macropexophagy and nitrogen limitation-induced microautophagy. In atg21 cells subjected to macropexophagy conditions, sequestration of peroxisomes tagged for degradation is initiated but fails to complete.     

A hexose transporter homologue controls glucose repression in the methylotrophic yeast Hansenula polymorpha

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser(85)) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.     

Proteomic analysis of extracellular proteins from Escherichia coli W3110

While numerous proteomic analyses have been carried out on Escherichia coli, the vast majority have focused on expression of intracellular proteins. Yet, recent literature reports imply that even in laboratory strains, significant proteins may be found outside the cell. Here, we identify extracellular proteins associated with nonpathogenic E. coli strain W3110. Two-dimensional gel electrophoresis (2DE) revealed approximately 66 prominent protein spots during exponential growth (4 and 8 h shake flask culture) in minimal medium. The absence of detectable nucleic acids in the culture supernatant implies these proteins did not result from cell lysis. MALDI-TOF MS was used to identify 44 proteins, most of which have been previously identified as either outer membrane or extracellular proteins. In addition, 2DE protease zymogram analysis was carried out which facilitated identification of three extracellular proteases, one of which was not observed during standard 2DE. Our results are consistent with previous findings which imply outer membrane proteins are shed during growth.     

Distribution and ecology of <Emphasis Type="Italic">Hemimysis anomala</Emphasis>, the latest invader of the Great Lakes basin

Since 2006, the known distribution of Hemimysis anomala has greatly expanded in the Great Lakes ecosystem, with, to date, 45 sites of occurrence among 91 monitored sites, located in four of the Great Lakes and the upper St. Lawrence River. By means of carbon and nitrogen stable isotopes, a first assessment of the feeding ecology of Hemimysis was completed. The δ¹³C values of 18 individuals collected in Lake Erie (Port Mainland) on a single date (Sept. 23, 2008) ranged from −30.2 to −24.5‰, indicating that Hemimysis could feed on multiple carbon sources including pelagic and littoral autochthonous and terrestrial carbon. In Lake Erie, variation in δ¹³C was related to δ¹⁵N, indicating the importance of food source for determining the trophic position of Hemimysis. The δ¹⁵N signatures of individuals were strongly related to their C/N ratios, suggesting that variations in the nutritional value of Hemimysis may depend on trophic position. Isotopic variation among individuals in Lake Erie was complemented by temporal variation in Lake Ontario. Monthly changes (from June to December 2008) in carbon isotope signatures were observed and related to changes in water temperature, highlighting the variations in the baseline prey signatures that fuel Hemimysis diets. The observed variation in stable isotope signatures occurring among individuals within a localized Hemimysis assemblage and temporally should be considered as a key design feature in further studies attempting to identify the possible effects of Hemimysis on nearshore food webs in the Great Lakes.     

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-beta was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-kappaB activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.     

ABA regulation of K(+)-permeable channels in maize subsidiary cells

An antiparallel-directed potassium transport between subsidiary cells and guard cells which form the graminean stomatal complex has been proposed to drive stomatal movements in maize. To gain insights into the coordinated shuttling of K(+) ions between these cell types during stomatal closure, the effect of ABA on the time-dependent K(+) uptake and K(+) release channels as well as on the instantaneously activating non-selective cation channels (MgC) was examined in subsidiary cells. Patch-clamp studies revealed that ABA did not affect the MgC channels but differentially regulated the time-dependent K(+) channels. ABA caused a pronounced rise in time-dependent outward-rectifying K(+) currents (K(out)) at alkaline pH and decreased inward-rectifying K(+) currents (K(in)) in a Ca(2+)-dependent manner. Our results show that the ABA-induced changes in time-dependent K(in) and K(out) currents from subsidiary cells are very similar to those previously described for guard cells. Thus, the direction of K(+) transport in subsidiary cells and guard cells during ABA-induced closure does not seem to be grounded solely on the cell type-specific ABA regulation of K(+) channels.     

30-year progress of membrane transport in plants

In the past 30 years enormous progress was made in plant membrane biology and transport physiology, a fact reflected in the appearance of textbooks. The first book dedicated to ‘Membrane Transport in Plants’ was published on the occasion of the ‘International Workshop on Membrane Transport in Plants’ held at the Nuclear Research Center, Jülich, Germany [Zimmermann and Dainty (eds) 1974] and was followed in 1976 by a related volume ‘Transport in plants II’ in the ‘Encyclopedia of plant physiology’ [Lüttge and Pitman (eds) 1976]. A broad spectrum of topics including thermodynamics of transport processes, water relations, primary reactions of photosynthesis, as well as more conventional aspects of membrane transport was presented. The aim of the editors of the first book was to bring advanced thermodynamical concepts to the attention of biologists and to show physical chemists and biophysicist what the more complex biological systems were like. To bundle known data on membrane transport in plants and relevant fields for mutual understanding, interdisciplinary research and clarification of problems were considered highly important for further progress in this scientific area of plant physiology. The present review will critically evaluate the progress in research in membrane transport in plants that was achieved during the past. How did ‘Membrane Transport in Plants’ progress within the 30 years between the publication of the first book about this topic (Zimmermann and Dainty 1974), a recent one with the same title (Blatt 2004), and today?