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Protein synthesis, processing, and trafficking
No full textProteins are the final executers of the genetic program of a cell and are responsible for the remarkable diversity in cell specialization that is typical of metazoan organisms. In order to perform this role, after synthesis, proteins need to be properly folded, assembled into oligomeric complexes and transported to their final destinations. In many cases protein folding and processing are coupled with protein trafficking so that the targeting process is unidirectional and irreversibly. Eukaryotic cells contain membrane-bound compartments, termed organelles, such as the mitochondria, the endoplasmic reticulum, the peroxisomes and the nucleus. Each organelle serves a particular purpose. The capacity of any organelle to fulfill its role in intracellular physiology depends on a characteristic set of protein components. This chapter briefly describes how proteins are synthesized and then focuses on their processing and delivery to their appropriate destinations within the cell. An understanding of the machines that catalyze protein folding, assembly, and targeting is relevant to the study of hematology providing a basis for an explanation of how malfunctions in these processes can cause blood disorders
The PHR Family: The Role of Extracellular Transglycosylases in Shaping Candida albicans Cells
Get PDFCandida albicans is an opportunistic microorganism that can become a pathogen causing mild superficial mycosis or more severe invasive infections that can be life-threatening for debilitated patients. In the etiology of invasive infections, key factors are the adaptability of C. albicans to the different niches of the human body and the transition from a yeast form to hypha. Hyphal morphology confers high adhesiveness to the host cells, as well as the ability to penetrate into organs. The cell wall plays a crucial role in the morphological changes C. albicans undergoes in response to specific environmental cues. Among the different categories of enzymes involved in the formation of the fungal cell wall, the GH72 family of transglycosylases plays an important assembly role. These enzymes cut and religate β-(1,3)-glucan, the major determinant of cell shape. In C. albicans, the PHR family encodes GH72 enzymes, some of which work in specific environmental conditions. In this review, we will summarize the work from the initial discovery of PHR genes to the study of the pH-dependent expression of PHR1 and PHR2, from the characterization of the gene products to the recent findings concerning the stress response generated by the lack of GH72 activity in C. albicans hyphae
Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 protein into the chitin ring and septum is required for proper morphogenesis in yeast
Get PDFGas1p is a glucan-elongase that plays a crucial role in yeast morphogenesis. It is predominantly anchored to the plasma membrane through a glycosylphosphatidylinositol but a fraction was also found covalently bound to the cell wall. We have used fusions with the green or red fluorescent proteins (GFP or RFP) to determine its localization. Gas1p was present in microdomains of the plasma membrane, at the mother-bud neck and in the bud scars. By exploiting the instability of RFP-Gas1p we identified mobile and immobile pools of Gas1p. Moreover, in chs3 cells the chitin ring and the cross-linked Gas1p were missing but this unveiled an additional unexpected localization of Gas1p along the septum line in cells at cytokinesis. Localization of Gas1p was also perturbed in a chs2 mutant where a remedial septum is produced. Phenotypic analysis of cells expressing a fusion of Gas1p to a trans-membrane domain unmasked new roles of the cell wall-bound Gas1p in the maintenance of the bud neck size and in cell separation. We present evidence that the Crh1p and Crh2p are required for tethering Gas1p to the chitin ring and bud scar. These results reveal a new mechanism of protein immobilization at specific sites of the cell envelop
GAS2 and GAS4 : a pair of developmentally regulated genes involved in spore wall assembly in yeast
No full textThe GAS multigene family comprehends 5 paralogs encoding GPI-anchored proteins endowed with glucanosyltransferase activity. Gas1p, the best characterized protein of the family, is expressed during vegetative growth and plays a crucial role in cell wall assembly. In order to study the role of GAS2 and GAS4 genes, that were previously classified as middle meiotic genes, we analysed their expression profiles during the entire sporulation process by Quantitative RT-PCR in a SK1 strain. GAS2 and GAS4 expression profiles were periodic and overlapping. A peak occurred at 7 h from induction of sporulation and at 10 h the mRNA levels were already decreased. Immunoblot analysis using polyclonal antibodies raised against the purified Gas4p revealed that Gas4p is a GPI-anchored 60 kDa membrane protein that is absent at the moment of induction of sporulation and reaches a maximum at 11 h from transfer of the cells to sporulation medium. The double null mutant gas2 gas4/ gas2 gas4 showed a severe reduction of the efficiency of sporulation whereas the single null mutants, gas2/gas2 and gas4/gas4 were similar to the isogenic strain. These results indicate that GAS2 and GAS4 are redundant genes which play partially overlapping functions in spore morphogenesis. To determine if meiosis was affected in the double null mutant, we monitored the kinetics of nuclei formation. Meiotic progression occurred normally when Gas2 and Gas4 proteins were absent A detailed analysis of the spore wall defects was performed using different kinds of assays. Spores of the double null mutant resulted to be morphologically aberrant, less viable, highly permeable to Zymolyase and Calcofluor. TEM and SEM analysis of the spores of the double mutant indicated that the glucan and chitosan layers are dissociated and an abnormal accumulation of spore wall material occurs. These results suggests that Gas2 and Gas4 proteins play a crucial role in spore morphogenesis by direecting the attachment of the inner and outer spore wall layers to each other
GAS2 and GAS4, a pair of developmentally regulated genes required for spore wall assembly in Saccharomyces cerevisiae
Get PDFThe GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a beta(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral
Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding
No full textGas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be beta-(1,3)-glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys-rich domain; and a highly O-glycosylated serine-rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of approximate to 10 mg.L-1 of medium, and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 x His tag. Purified soluble Gas1 protein showed beta-(1,3)-glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane-bound Gas1p were analyzed in S. cerevisiae. The Gas1-C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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