Folding-competent and Folding-defective Forms of Ricin A Chain Have Different Fates after Retrotranslocation from the Endoplasmic Reticulum

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTA_Δ), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER–Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTA_Δ, although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.     

Do rural impoundments in coastal Bay of Fundy,Canada sustain adequate habitat for wildlife?

In Canada and elsewhere in North America, impoundments are created in compensation for historic wetland loss and for habitat loss due to development projects, but these new sites are infrequently evaluated to determine how effectively they function. The Cumberland Marsh Region (CMR), located at the head of the Bay of Fundy, Canada, is of importance to migratory birds and has been subject to 300+ years of anthropogenic alteration, including impoundment creation on diked and drained tidal marsh in the last five decades. Wetland managers have noticed a pervasive decline in impoundment productivity leading to reduced waterbird usage (senescence). To understand factors that promote senescence, we analyzed abiotic and biotic proxies in sediment archives from six freshwater impoundments in two coastal watersheds to assess spatial trends across the CMR within recent decades. Our results demonstrate that impoundment productivity is driven by autochthonous nutrient sources (C/N between 7.7 and 14.4), but biogeochemical conditions can be highly variable among impoundments despite their proximity. Biogeochemical variation among top-of-core sediment samples from each impoundment was generally minimal, and thus we believe that the aging of impoundments has resulted in low productivity and organic matter accumulation due to dike stabilization and declines in nutrient loading. We conclude that these freshwater impoundments (in the CMR and likely other similar settings) are not highly productive, and may not provide abundant forage and optimal wildlife habitat which is expected of these systems; adaptive management strategies and hydrologic rehabilitation merit consideration to enhance ecological functioning. Understanding landscape attributes, hydrologic dynamics, and conditions prior to and after major human alterations should be a priority in future compensation projects.     

Allopolyploid speciation of the Mexican tetraploid potato species Solanum stoloniferum and S. hjertingii revealed by genomic in situ hybridization

Thirty-six percent of the wild potato (Solanum L. section Petota Dumort.) species are polyploid, and about half of the polyploids are tetraploid species (2n = 4x = 48). Determination of the type of polyploidy and development of the genome concept for members of section Petota traditionally has been based on the analysis of chromosome pairing in species and their hybrids and, most recently, DNA sequence phylogenetics. Based on these data, the genome designation AABB was proposed for Mexican tetraploid species of series Longipedicellata Buk. We investigated this hypothesis with genomic in situ hybridization (GISH) for both representatives of the series, S. stoloniferum Schltdl. and S. hjertingii Hawkes. GISH analysis supports an AABB genome constitution for these species, with S. verrucosum Schltdl. (or its progenitor) supported as the A genome donor and another North or Central American diploid species (S. cardiophyllum Lindl., S. ehrenbergii (Bitter) Rydb., or S. jamesii Torrey) as the B genome donor. GISH analysis of chromosome pairing of S. stoloniferum also confirms the strict allopolyploid nature of this species. In addition, fluorescence in situ hybridization data suggest that 45S rDNA regions of the two genomes of S. stoloniferum were changed during coevolution of A and B genomes of this allotetraploid species.     

Corticioid fungi from the Kimberley Region,Western Australia

Summary  Corticioid fungi from the Kimberley Region of Western Australia are reviewed. 31 species are reported, of which five, Aleurodiscus kimberleyanus, Athelopsis vesicularis, Dendrothele cornivesiculosa, Hyphoderma tubulicystidium, and Phanerochaete subcrassispora are described as new. Grandinia glauca is given the new combination Grammothele glauca, and Hydnum investiens the new combination Phanerochaete investiens. A further eight species are recorded which have not previously been reported from Australia.     

A Human Embryonic Kidney 293T Cell Line Mutated at the Golgi ��-Mannosidase II Locus

Disruption of Golgi α-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi α-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi α-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.Mammalian N-linked glycosylation is characterized by significant chemical heterogeneity generated by an array of competing glycosidases and glycosyltransferases (1). The structural analysis of recombinant glycoproteins, such as human erythropoietin (2, 3), has illustrated the capacity of mammalian expression systems for generating diverse N-linked glycans.Heterogeneity develops during egress of a glycoprotein through the secretory system (1). N-linked glycosylation is initiated in the rough endoplasmic reticulum (ER)⁴ by the co-translational transfer of Glc₃Man₉GlcNAc₂ to the asparagine residues of the glycosylation sequon. In the absence of protein misfolding, hydrolysis by ER α-mannosidase I plus α-glucosidase I and II results in the transfer of glycoproteins dominated by the D1,D3 isomer of Man₈GlcNAc₂ glycans to the Golgi apparatus (4). Further processing by Golgi α-mannosidases IA–C generates Man₅GlcNAc₂ (5–7), the principle substrate for UDP-N-acetyl-d-glucosamine:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I (GnT I). The action of this enzyme yields classic hybrid-type glycans with mannosyl 6-antennae and processed 3-antennae (1). In the absence of the GnT III-mediated addition of bisecting GlcNAc, the two terminal α-mannose residues of the 6-antenna of hybrid-type glycans are cleaved by Golgi α-mannosidase II, forming mono-antennary complex-type glycans. These may then be processed by N-acetylglucosaminyltransferases, generating multiantennary complex-type glycans of enormous potential heterogeneity following the sequential transfer of monosaccharides such as galactose, N-acetylgalactosamine, fucose, and N-acetylneuraminic acid (8).The importance of this carbohydrate diversity in metazoan biology is illustrated by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. In humans, about 12 congenital disorders of glycosylation (CDG) have been identified with defects in the biosynthesis of N-linked glycans (9). One disorder characterized by changes in glycosylation is congenital dyserythropoietic anemia type II (hereditary erythroblastic multinuclearity with a positive acidified serum lysis test (HEMPAS)) (10, 11). HEMPAS is a heterogenous autosomal recessive disorder that renders erythrocytes prone to lysis. Although the precise molecular basis of HEMPAS remains to be determined, it is characterized by either a reduction in β1→4-galactosyltransferase, GnT II, or, in some patients, Golgi α-mannosidase II activity (11, 12). Interestingly, the increase in cell surface terminal mannose in mice deficient in Golgi α-mannosidase II leads to autoimmunity through chronic activation of the innate immune system (13, 14).Lectin-resistant (Lec) cell lines harboring loss- or gain-of-function mutations affecting the biosynthesis of N-glycans have emerged as powerful tools for the investigation of these disorders (15). For example, genetic complementation using Lec2, containing a mutation in the cytosine monophosphate sialic acid transporter, was used to identify a novel CDG, type IIf (16). Lectin-resistant cell lines can also be used as hosts to study naturally occurring mutations, as in the case of CHO Lec23 cells used to screen α-glucosidase I mutations in CDG, type IIb (17). Other applications of lectin-resistant cell lines include the expression of specific glycoforms of therapeutic glycoproteins. Manipulating the structure of their carbohydrate moieties modulates the pharmacological properties of glycoproteins by altering their bioactivity, serum half-life, and/or tissue tropism (18). For example, β-glucocerebrosidase expressed in CHO Lec1 cells (deficient in GnT I activity) exhibits mannosylation and improved macrophage uptake for the treatment of Gaucher disease (19). Lectin-resistant CHO cell lines have also been used to improve the crystallizability of glycoproteins for structural determination by x-ray crystallography (20–24).The expression of therapeutic glycoproteins as one or more defined “glycoforms” is essential for their optimization and may even be necessary to obtain regulatory approval (25). To this end, eukaryotic expression systems have been developed that allow glycosylation to be controlled. Recently, Pichia pastoris-based strains with human glycosyltransferases have been established, allowing the expression of glycoforms with oligomannose-, hybrid-, and some complex-type glycans (26, 27) and even sialylated complex-type structures (28). However, mammalian expression remains the dominant technology in industrial settings, presumably because of its reliability for the expression of human secreted glycoproteins.Although the majority of lectin-resistant cell lines have been generated using CHO cells, no Golgi α-mannosidase II-deficient CHO cell line has been generated thus far (15). Furthermore, only one human lectin-resistant cell line, i.e. GnT I-deficient (Lec1) HEK 293S cells (29), has been produced. Hybrid-type glycosylation has been reported to accumulate in ricin-resistant baby hamster kidney cells (30, 31); however, these cells contain a reduced but detectable level of cell-surface complex-type glycans, consistent with an incomplete ablation of Golgi α-mannosidase II activity (31). Moreover, the hybrids from one of these lines contain a trimannosyl rather than pentamannosyl core and appear to be heavily influenced by GnT II deficiency, resulting in the formation of what are now commonly called monoantennary complex-type glycans (32–34). We now describe the isolation of an HEK 293T cell line mutated at the MAN2A1 locus and deficient in Golgi α-mannosidase II activity via selection with ricin.