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      Biological functions of the autophagy-related proteins Atg4 and Atg8 in Cryptococcus neoformans

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          Abstract

          Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related ( ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions.

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          Most cited references57

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          A ubiquitin-like system mediates protein lipidation.

          Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.
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            High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.

            A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.
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              A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

              SD Emr (1995)
              We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.
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                Author and article information

                Contributors
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: SoftwareRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 April 2020
                2020
                : 15
                : 4
                : e0230981
                Affiliations
                [1 ] Departamento de Ciências Biológicas, Universidade Federal de São Paulo, Diadema, São Paulo, Brazil
                [2 ] Departamento de Infectologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
                [3 ] School of BioSciences, University of Melbourne, Melbourne, Victoria, Australia
                Faculty of Medicine, University of Belgrade, SERBIA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-6929-0566
                Article
                PONE-D-19-32465
                10.1371/journal.pone.0230981
                7135279
                32251488
                4c12b40a-abb8-46bd-be4d-62e3fe340004
                © 2020 Roberto et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 November 2019
                : 12 March 2020
                Page count
                Figures: 6, Tables: 0, Pages: 23
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001807, Fundação de Amparo à Pesquisa do Estado de São Paulo;
                Award ID: 2015/04400-9
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001807, Fundação de Amparo à Pesquisa do Estado de São Paulo;
                Award ID: 2016/50185-5
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001807, Fundação de Amparo à Pesquisa do Estado de São Paulo;
                Award ID: 2016/14542-8
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002322, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior;
                Award ID: 001
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002322, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior;
                Award ID: 88881.133481/2016-01
                Award Recipient :
                This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants #2015/04400-9, #2016/50185-5 to MAV and #2016/14542-8 to RCP, by the SPRINT-University of Melbourne travel grant to AI, and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) grants #001 and #88881.133481/2016-01 to TNR.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Cryptococcus
                Cryptococcus Neoformans
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Fungal Pathogens
                Cryptococcus Neoformans
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Fungal Pathogens
                Cryptococcus Neoformans
                Biology and Life Sciences
                Mycology
                Fungal Pathogens
                Cryptococcus Neoformans
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Death
                Autophagic Cell Death
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Model Organisms
                Saccharomyces Cerevisiae
                Research and Analysis Methods
                Model Organisms
                Saccharomyces Cerevisiae
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Yeast
                Saccharomyces
                Saccharomyces Cerevisiae
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Yeast and Fungal Models
                Saccharomyces Cerevisiae
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vacuoles
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Research and Analysis Methods
                Computational Techniques
                Split-Decomposition Method
                Multiple Alignment Calculation
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Alignment
                Biology and Life Sciences
                Genetics
                Fungal Genetics
                Biology and Life Sciences
                Mycology
                Fungal Genetics
                Custom metadata
                All relevant data are within the manuscript and its Supporting Information files.

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