Analysis of the role of actin in endocytosis and osmoregulationin Saccharomyces cerevisiae

Persistent Link:
http://hdl.handle.net/10150/284065
Title:
Analysis of the role of actin in endocytosis and osmoregulationin Saccharomyces cerevisiae
Author:
ODell, Johanna Lynn
Issue Date:
1999
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The actin cytoskeleton is a protein complex composed of actin and actin-associated proteins that regulate actin assembly and function. Actin function is required for a variety of cellular processes including locomotion, secretion, endocytosis and organelle movement. Although actin function is implicated in these processes we have yet to determine whether actin is a direct participant or simply required for general cell health. The work presented here examines actin's requirement during osmoregulation and endocytosis in Saccharomyces cerevisiae . Analysis of the phenotypes exhibited in a collection of isogenic actin mutants reveals correlations between the phenotypes of sporulation, endocytosis and salt sensitivity. We propose a causal relationship and demonstrate actin mutants may be salt sensitive because salt inhibits endocytosis, exacerbating endocytosis defects caused by mutant actin. Mapping the salt sensitive and endocytosis defective alleles on the atomic structure of actin reveals that the actin-Sac6p interaction is crucial for salt resistance and efficient endocytosis. Furthermore, our studies indicate that decreased endocytosis rates in NaCl act independently of clathrin and the high-osmolarity glycerol (HOG) signal transduction pathway. We quantitatively examined whether actin directly participates in the trafficking of plasma membrane proteins by examining defects in cells treated with the actin inhibitor Latrunculin-A (Lat-A). By measuring protein degradation, we demonstrate that actin disruption results in turnover defects of both the receptor, Ste2p and the non-receptor, Ste6p. However, actin disruption more severely alters receptor degradation. We suggest that actin plays a direct role in Ste2p turnover and an indirect role in Ste6p turnover. We further quantitatively compared actin's contribution to constitutive versus receptor-mediated turnover by analyzing Ste2p degradation in the presence or absence of alpha-factor and determined that actin disruption more severely affected Ste2p turnover in the absence of ligand. Using electron microscopy, we examined the interactions between actin and two actin-bundling proteins, fimbrin and Ef1alpha. We analyzed the ability of mutant actins to form filaments and assayed which alleles exhibited defective Sac6p interactions. Our analysis of the interactions between different combinations of mutant and wild-type actin and Sac6p demonstrate that the in vivo suppression between actin and Sac6p occurs by restoring binding and bundling activity.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.; Biology, Genetics.; Biology, Cell.; Biology, Microbiology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Molecular and Cellular Biology
Degree Grantor:
University of Arizona
Advisor:
Adams, Alison

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleAnalysis of the role of actin in endocytosis and osmoregulationin Saccharomyces cerevisiaeen_US
dc.creatorODell, Johanna Lynnen_US
dc.contributor.authorODell, Johanna Lynnen_US
dc.date.issued1999en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe actin cytoskeleton is a protein complex composed of actin and actin-associated proteins that regulate actin assembly and function. Actin function is required for a variety of cellular processes including locomotion, secretion, endocytosis and organelle movement. Although actin function is implicated in these processes we have yet to determine whether actin is a direct participant or simply required for general cell health. The work presented here examines actin's requirement during osmoregulation and endocytosis in Saccharomyces cerevisiae . Analysis of the phenotypes exhibited in a collection of isogenic actin mutants reveals correlations between the phenotypes of sporulation, endocytosis and salt sensitivity. We propose a causal relationship and demonstrate actin mutants may be salt sensitive because salt inhibits endocytosis, exacerbating endocytosis defects caused by mutant actin. Mapping the salt sensitive and endocytosis defective alleles on the atomic structure of actin reveals that the actin-Sac6p interaction is crucial for salt resistance and efficient endocytosis. Furthermore, our studies indicate that decreased endocytosis rates in NaCl act independently of clathrin and the high-osmolarity glycerol (HOG) signal transduction pathway. We quantitatively examined whether actin directly participates in the trafficking of plasma membrane proteins by examining defects in cells treated with the actin inhibitor Latrunculin-A (Lat-A). By measuring protein degradation, we demonstrate that actin disruption results in turnover defects of both the receptor, Ste2p and the non-receptor, Ste6p. However, actin disruption more severely alters receptor degradation. We suggest that actin plays a direct role in Ste2p turnover and an indirect role in Ste6p turnover. We further quantitatively compared actin's contribution to constitutive versus receptor-mediated turnover by analyzing Ste2p degradation in the presence or absence of alpha-factor and determined that actin disruption more severely affected Ste2p turnover in the absence of ligand. Using electron microscopy, we examined the interactions between actin and two actin-bundling proteins, fimbrin and Ef1alpha. We analyzed the ability of mutant actins to form filaments and assayed which alleles exhibited defective Sac6p interactions. Our analysis of the interactions between different combinations of mutant and wild-type actin and Sac6p demonstrate that the in vivo suppression between actin and Sac6p occurs by restoring binding and bundling activity.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
dc.subjectBiology, Genetics.en_US
dc.subjectBiology, Cell.en_US
dc.subjectBiology, Microbiology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMolecular and Cellular Biologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorAdams, Alisonen_US
dc.identifier.proquest9960268en_US
dc.identifier.bibrecord.b40274846en_US
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