A Study Of Vacuolar Kinase Env7 Trafficking In Saccharomyces Cerevisiae
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A Study Of Vacuolar Kinase Env7 Trafficking In Saccharomyces Cerevisiae
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Author : Leah Frisardi
language : en
Publisher:
Release Date : 2019
A Study Of Vacuolar Kinase Env7 Trafficking In Saccharomyces Cerevisiae written by Leah Frisardi and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2019 with Autophagic vacuoles categories.
Abstract: The vacuole in Saccharomyces cerevisiae (baker’s yeast) is functionally analogous to the human lysosome. Endosome and vacuolar interface 7 (Env7) has been recently identified in the Gharakhanian laboratory as a protein kinase that regulates vacuolar fusion and has a human ortholog, STK16. Previous research in our laboratory has shown that Env7 localizes to the vacuolar membrane in yeast. Proteins destined to function at the yeast vacuole are transported from the Golgi via two distinct pathways: the carboxypeptidase Y (CPY) pathway and the alkaline phosphatase (ALP) pathway. The CPY pathway requires an endosomal intermediate while the ALP pathway requires an adaptor protein complex (AP-3). The CPY pathway serves as the default pathway for the vast majority of proteins moving from the Golgi to the vacuole. Only 9 proteins are known to be transported via the ALP pathway in yeast, all of which are vacuolar membrane proteins and one of which is Yck3, a palmitoylated protein kinase with a similar function of Env7. Proteins normally transported to the yeast vacuole via the ALP pathway are able to partially revert to the CPY pathway when AP-3 function is disrupted. In the absence of a functional CPY pathway, however, the AP-3 pathway remains cargo-specific. Here we show that when the CPY pathway is disrupted in yeast cells, Env7 shows a significant increase in mislocalization. When the ALP pathway is disrupted, Env7 shows no significant mislocalization. Proteins transported via the ALP pathway interact with AP-3 via either an acidic-dileucine-like or a tyrosine-based adaptin sorting signal. Analysis of the amino acid sequence of Env7 showed one potential acidic-dileucine like signal and four potential tyrosine-based adaptin sorting signals. Mutagenesis of two of these sorting signals caused no significant mislocalization of Env7 in yeast. These results implicate the CPY pathway in the vacuolar trafficking of Env7 in yeast.
Trafficking Of Protein Kinase Env7 To The Lysosomal Vacuole Of Saccharomyces Cerevisiae
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Author : Maria Carmen Ramos (Graduate student)
language : en
Publisher:
Release Date : 2022
Trafficking Of Protein Kinase Env7 To The Lysosomal Vacuole Of Saccharomyces Cerevisiae written by Maria Carmen Ramos (Graduate student) and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2022 with Protein kinases categories.
Abstract: The Saccharomyces cerevisiae protein kinase Env7, which negatively regulates homotypic vacuolar membrane fusion during cell stress and budding, has been shown to localize to the yeast vacuole under normal wildtype conditions. Proteins destined for the yeast vacuole traffic either directly from the trans-Golgi network via the AP-3 complex (ALP pathway) or through an endosomal-intermediate (CPY pathway). This study furthers our understanding of Env7 localization to supplement results previously presented in our lab. Here, a time-wise inactivation of the CPY pathway probes at the possibility that Env7 is first mislocalized to or retained at the Golgi. In persistently defective CPY pathway mutants, Env7 protein is further degraded. Single-point mutations of AP-3 complex sorting signal motifs identified within Env7 are deemed unnecessary for the efficient transport to the yeast vacuole. Ultimately, a functional CPY pathway plays an important role in normal Env7 localization at the vacuole, while the ALP pathway does not. Furthermore, an intact CPY pathway is essential for the persistent presence of Env7 in cells, and presumably Env7 stability.
Studies On Saccharomyces Cerevisiae Vacuolar Membrane Kinase Env7
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Author : Sara Patrice Valencia
language : en
Publisher:
Release Date : 2018
Studies On Saccharomyces Cerevisiae Vacuolar Membrane Kinase Env7 written by Sara Patrice Valencia and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2018 with Biological transport categories.
Abstract: The yeast vacuole is a dynamic organelle that is functionally analogous to the mammalian lysosome and serves as a model for the study of membrane fusion and fission. Mechanisms of membrane fission and fusion dynamics have been well conserved from yeast to humans. However, the regulatory mechanisms that govern cellular fission and fusion dynamics remain poorly understood. Our lab has previously established that Env7 is a conserved yeast palmitoylated protein kinase that localizes to the yeast vacuole and negatively regulates vacuole membrane fusion during budding and hyperosmotic stress. Phosphorylation of Env7 is dependent on another vacuolar membrane kinase, Yck3, and is essential to Env7 stability and negative regulation of vacuolar membrane fusion. In this study, we aim to further our understanding of the role Env7 plays at the vacuole by 1) characterizing the phosphorylation of Env7 as a function of cell cycle using cell cycle arrest and synchronization techniques, and 2) generating functional biochemically tagged Yck3 to be used in interaction and phosphorylation assays with Env7. Cell cycle arrest and synchronization techniques have not previously been established in our lab. Here, we report reliable protocols of inducing cell cycle arrest using [alpha]-factor mating pheromone and Hydroxyurea. Results show that Env7 is hyperphosphorylated when cell cycle is arrested at G1 phase using [alpha]-factor mating pheromone. In both cell cycle arrest approaches, vacuoles show significant increase in fragmentation, and Env7 remains localized to the membrane of fragmented vacuoles. In cell culture synchronized with [alpha]-factor, Env7 shows an increase in phosphorylation between S-phase and G2, with decreased phosphorylation in M and G1. We were successful in engineering biochemically tagged Yck3 and established that the expressed 6XHis-Yck3 is functional and able to restore phosphorylation of Env7 in vivo. We also established that overexpressed 6XHis-Yck3 localized correctly to the vacuolar membrane. These tools will be used in future studies on interactions and regulation of membrane fusion.
Investigations Of The Functional Interface Between Env7 And Torc1 In Saccharomyces Cerevisiae
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Author : Jonathan Shaoulian
language : en
Publisher:
Release Date : 2016
Investigations Of The Functional Interface Between Env7 And Torc1 In Saccharomyces Cerevisiae written by Jonathan Shaoulian and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2016 with Autophagic vacuoles categories.
Abstract: In eukaryotic cells, deficiencies in proteins involved in lysosomal trafficking and function result in serious human disorders like Tay-Sach’s Disease or Alzheimer’s. The vacuole in Saccharomyces cerevisiae serves as a model for the mammalian lysosome. Our lab has identified ENV7, a novel gene involved in the late-endosome-to vacuole interface in Saccharomyces cerevisae. ENV7 (an ortholog of human STK16) encodes Env7, a vacuolar membrane kinase that negatively regulates vacuolar fusion. Tor1 is another conserved vacuolar membrane kinase, and has been proposed as a vacuolar fragmentation factor. Tor1, or its homolog Tor2, are the functional kinases of Tor Complex 1 (TORC1). Activated TORC1 stimulates cell growth and proliferation, as well as vacuolar fragmentation. In mammals, hyperactivity of TORC1 plays a major role in cancers and diabetes. Conversely, inhibition of TORC1 by starvation or treatment by the drug rapamycin, is associated with the initiation of autophagy, which itself involves a number of fusion events at the vacuole. Lastly, it has been shown that defects at various stages of vesicular trafficking play a role in disrupting Tor1 transport to the vacuole and perturbing the function of TORC1. Therefore, I hypothesized that there is a functional interface between Env7 and TORC1. Here I report that endogenously expressed Env7-GFP is localized to the vacuolar membrane, and upon inhibition of TORC1 by rapamycin, there is a significant mislocalization of Env7-GFP to diffuse cytoplasmic patterns. Overexpressed GFP-Env7 is also mislocalized when TORC1 activity is downregulated, but the mislocalization is to Golgi structures and to the plasma membrane. Additionally, while the absence of Tor1 is not sufficient to affect the localization of overexpressed GFP-Env7, when TORC1 is compromised by a deletion of TCO89 or by a more potent inhibition by rapamycin in both tor1[delta] and tco89[delta] cells, there is a significant mislocalization of GFP-Env7 to the plasma membrane. Taken together, my findings demonstrate that the mislocalization of Env7 is dependent on both the level of expression of Env7 and the state of TORC1 activity. Therefore, the results thus far support a possible functional interface between Env7 and TORC1. Furthermore, when TORC1 is inhibited by rapamycin treatment, unlike Env7-GFP, endogenously expressed Tor1-GFP remains on the vacuole, but forms numerous clusters at this location. In the course of testing my hypothesis, the effects of TORC1 inhibition on vacuolar morphology and onset of autophagy were also noted. I report that tor1[delta] cells, when treated by rapamycin, display a higher percentage of enlarged vacuole morphology relative to wt, and that this enlarged vacuole morphology seems to be associated with slightly increased levels of autophagy. This suggests that the absence of Tor1, and presumably its substitution by Tor2, compromises TORC1 and makes the cell more sensitive to rapamycin-induced autophagy. Additionally, I also report that absence of Env7 has no significant effect on vacuolar enlargement and the onset of autophagy in response to rapamycin treatment, and thus, does not affect the autophagy regulation function of TORC1. The results of this study suggest a functional interface between Env7 and TORC1. Since both are conserved in humans, such an interface may also be conserved in humans.
Env7p Localizes To The Vacuole Membrane And Plays A Role In Vacuolar Protein Processing
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Author : Maribeth Serranilla
language : en
Publisher:
Release Date : 2012
Env7p Localizes To The Vacuole Membrane And Plays A Role In Vacuolar Protein Processing written by Maribeth Serranilla and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2012 with Lysosomal storage diseases categories.
Abstract: The vacuole of the yeast Saccharomyces cerevisiae functions comparably to the mammalian lysosome. Its functions include protein degradation, pH/ion homeostasis, down regulation of cell surface receptors, and the ability to adapt to different environmental stresses. Defects in lysosomal trafficking are implicated with neurodegenerative diseases; therefore, understanding vacuolar trafficking will lead to more insight into lysosomal trafficking. Through our genome wide screen to identify mutants defective in the late stages of vacuolar trafficking, our lab has uncovered several previously uncharacterized ENdosome and Vacuole interface (ENV) genes. This study is focused on the characterization of ENV7, a putative serine/threonine kinase that has 29% identity to the human STK16 kinase whose function is unknown. Complementation experiments show that ENV7 is responsible for correct processing of Carboxypeptidase Y. Overexpression of the gene results in no pleiotropic phenotypes within the parameters tested. Env7p localizes at the vacuolar membrane which led to further examination of the deletion mutant, env7[delta], in vacuolar events. Microscopic analyses of env7[delta] reveal no vacuolar morphology, bulk endocytic trafficking, and acidification function defects comparable to WT (BY4742). Growth assays reveal no fitness survival, and pH/ion homeostasis defects associated with env7[delta]. Overall, this study establishes that ENV7 is localized to the vacuolar membrane and is involved in vacuolar protein processing independent of vacuolar morphology and acidification functions.
Saccharomyces Advances In Research And Application 2013 Edition
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Author :
language : en
Publisher: ScholarlyEditions
Release Date : 2013-05-01
Saccharomyces Advances In Research And Application 2013 Edition written by and has been published by ScholarlyEditions this book supported file pdf, txt, epub, kindle and other format this book has been release on 2013-05-01 with Science categories.
Saccharomyces—Advances in Research and Application: 2013 Edition is a ScholarlyBrief™ that delivers timely, authoritative, comprehensive, and specialized information about ZZZAdditional Research in a concise format. The editors have built Saccharomyces—Advances in Research and Application: 2013 Edition on the vast information databases of ScholarlyNews.™ You can expect the information about ZZZAdditional Research in this book to be deeper than what you can access anywhere else, as well as consistently reliable, authoritative, informed, and relevant. The content of Saccharomyces—Advances in Research and Application: 2013 Edition has been produced by the world’s leading scientists, engineers, analysts, research institutions, and companies. All of the content is from peer-reviewed sources, and all of it is written, assembled, and edited by the editors at ScholarlyEditions™ and available exclusively from us. You now have a source you can cite with authority, confidence, and credibility. More information is available at http://www.ScholarlyEditions.com/.
Cytoskeleton Methods And Protocols
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Author : Ray H. Gavin
language : en
Publisher: Humana
Release Date : 2015-10-24
Cytoskeleton Methods And Protocols written by Ray H. Gavin and has been published by Humana this book supported file pdf, txt, epub, kindle and other format this book has been release on 2015-10-24 with Science categories.
The third edition of this volume focuses on experimental models that are useful for investigating various aspects of cytoskeleton structure and function. Animal, plant, protist, and fungal models highlight twenty-four chapters that provide detailed protocols for live and fixed-cell imaging, dynamics of cytoskeleton components, cell and organelle motility, and genetics and proteomics. Written in the highly successful Methods in Molecular Biology series format, protocols in each chapter are up-to-date menus organized in a useful step-by-step format appropriate for novice and established investigators. Each chapter is equipped with a valuable notes section that provides a troubleshooting guide and helpful, and often unpublished, technical information aimed at ensuring success with implementation of the protocols. Authoritative and thorough, Cytoskeleton Methods and Protocols, Third Edition helps researchers expand their understanding of cytoskeleton structure and function.
The Quest For Food
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Author : Harald Brüssow
language : en
Publisher: Springer Science & Business Media
Release Date : 2007-05-11
The Quest For Food written by Harald Brüssow and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2007-05-11 with Cooking categories.
This book explores the links between food and human cultural and physical evolution. Each chapter begins by summarizing the basic knowledge in the field, discusses recent research results, and confirms or challenges established concepts, inviting new insight and provoking new questions. This book catalyzes discussion between scientists working on one side in food science and on the other side in biological and biomedical research.
The Logic Of Chance
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Author : Eugene V. Koonin
language : en
Publisher: FT Press
Release Date : 2011-06-23
The Logic Of Chance written by Eugene V. Koonin and has been published by FT Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2011-06-23 with Science categories.
The Logic of Chance offers a reappraisal and a new synthesis of theories, concepts, and hypotheses on the key aspects of the evolution of life on earth in light of comparative genomics and systems biology. The author presents many specific examples from systems and comparative genomic analysis to begin to build a new, much more detailed, complex, and realistic picture of evolution. The book examines a broad range of topics in evolutionary biology including the inadequacy of natural selection and adaptation as the only or even the main mode of evolution; the key role of horizontal gene transfer in evolution and the consequent overhaul of the Tree of Life concept; the central, underappreciated evolutionary importance of viruses; the origin of eukaryotes as a result of endosymbiosis; the concomitant origin of cells and viruses on the primordial earth; universal dependences between genomic and molecular-phenomic variables; and the evolving landscape of constraints that shape the evolution of genomes and molecular phenomes. "Koonin's account of viral and pre-eukaryotic evolution is undoubtedly up-to-date. His "mega views" of evolution (given what was said above) and his cosmological musings, on the other hand, are interesting reading." Summing Up: Recommended Reprinted with permission from CHOICE, copyright by the American Library Association.
Candida And Candidiasis
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Author : Richard A. Calderone
language : en
Publisher: American Society for Microbiology Press
Release Date : 2011-12-07
Candida And Candidiasis written by Richard A. Calderone and has been published by American Society for Microbiology Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2011-12-07 with Medical categories.
The underlying mechanisms of Candida and candidiasis and promising new directions in drug discovery and treatment. • Reviews all aspects of this common fungal pathogen and its impact on human health, from the basic biology of Candida albicans to the clinical management of candidiasis. • Reviews the latest basic and clinical research, focusing on findings in genome variability, host-pathogen interactions, antifungal resistance and drug discovery, and diagnostics to foster better understanding and treatment of candidiasis. • Examines recent discoveries that have shed light on morphogenesis and the cell cycle, including how new findings on host responses may have applications for the diagnosis of blood-borne candidiasis.