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Intracellular trafficking of ricin A chain in relation to its cytotoxicity and depurination in Saccharomyces cerevisiae

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Title
Intracellular trafficking of ricin A chain in relation to its cytotoxicity and depurination in Saccharomyces cerevisiae
Name (type = personal)
NamePart (type = family)
Yan
NamePart (type = given)
Qing
DisplayForm
Qing Yan
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Tumer
NamePart (type = given)
Nilgun
DisplayForm
Nilgun Tumer
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
chair
Name (type = personal)
NamePart (type = family)
Leustek
NamePart (type = given)
Thomas
DisplayForm
Thomas Leustek
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Dong
NamePart (type = given)
Juan
DisplayForm
Juan Dong
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Li
NamePart (type = given)
Xiaoping
DisplayForm
Xiaoping Li
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Grant
NamePart (type = given)
Barth
DisplayForm
Barth Grant
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
outside member
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
Graduate School - New Brunswick
Role
RoleTerm (authority = RULIB)
school
TypeOfResource
Text
Genre (authority = marcgt)
theses
OriginInfo
DateCreated (qualifier = exact)
2013
DateOther (qualifier = exact); (type = degree)
2013-10
Place
PlaceTerm (type = code)
xx
Language
LanguageTerm (authority = ISO639-2b); (type = code)
eng
Abstract (type = abstract)
Ricin is a heterodimeric protein composed of ricin A chain (RTA) and ricin B chain (RTB). The extreme toxicity of ricin is mainly attributed to the enzymatically active RTA, which specifically removes an adenine residue from the universally conserved α-sarcin/ricin loop (SRL) in the 28S rRNA and inhibits protein synthesis. For RTA to exert its depurination activity, it has to undergo retrograde transport pathway to enter the cytosol. Therefore, the intracellular trafficking of RTA is an important aspect of ricin mediated cell death. The goal of the present study is to understand the structural features of RTA which contribute to its intracellular transport and the host factors involved in its trafficking in Saccharomyces cerevisiae. To visualize RTA, the enhanced green fluorescent protein (EGFP) is fused to mature RTA (matRTA-EGFP) containing 267-amino acid residues and precursor RTA (preRTA-EGFP) containing a 35-amino acid N-terminal extension followed by mature RTA. When preRTA-EGFP is expressed in the endoplasmic reticulum (ER) lumen of Saccharomyces cerevisiae, it follows two parallel pathways: 1) the well characterized ER-to-cytosol dislocation through the ER-associated degradation (ERAD) pathway, 2) vacuole transport after initial localization to the ER. In chapter two, I studied the roles of the important structural domains and sequence motifs of RTA in its trafficking and toxicity by mutational analysis. I showed that the 26-amino acid signal peptide within the N-terminal extension of preRTA is responsible for the ER targeting and the following nine-amino acid propeptide is important for glycosylation and efficient vacuole transport. The N-glycosylation of RTA promotes efficient ER export to the cytosol and vacuole, and contributes to the toxicity of RTA. The C-terminal hydrophobic domain of RTA is critical for transport out of the ER. In chapter three, I further investigated how the ER-to-vacuole transport and ER-to-cytosol dislocation pathways of preRTA affect its depurination and toxicity by using wild type preRTA and nontoxic preRTA mutants. The altered sequence motifs in preRTA lead to different translocation pathways. This provides the advantage of studying the contribution of their trafficking pathways to the toxicity of RTA. I present evidence that vacuole transport is an alternative degradation pathway of preRTA and contributes to the depurination reduction. The dislocated wild type preRTA and preRTA mutants have differential requirements for the cytosolic ERAD component peptide:N-glycanase (PNGase; yeast Png1), which is involved in the degradation of its substrates. In chapter four, the roles of yeast cellular components in the trafficking of RTA were studied to unravel the host genes in the ricin trafficking pathways. A genome wide screen of yeast nonessential gene knockout collection against RTA led to the discovery of genes in the Hrd1p complex and AP-2 complex, whose deletions conferred resistance to preRTA but not to matRTA. The components of the Hrd1p complex are responsible for the dislocation of preRTA. The Apl3p subunit of AP-2 complex is involved in the vacuole transport of preRTA. The study has provided important insights into the mechanism of intracellular trafficking of RTA and its role in toxicity.
Subject (authority = RUETD)
Topic
Plant Biology
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_5004
PhysicalDescription
Form (authority = gmd)
electronic resource
InternetMediaType
application/pdf
InternetMediaType
text/xml
Extent
xii, 163 p. : ill.
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
Note (type = vita)
Includes vita
Note (type = statement of responsibility)
by Qing Yan
Subject (authority = ETD-LCSH)
Topic
Ricin--Toxicology
Subject (authority = ETD-LCSH)
Topic
Saccharomyces cerevisiae
RelatedItem (type = host)
TitleInfo
Title
Graduate School - New Brunswick Electronic Theses and Dissertations
Identifier (type = local)
rucore19991600001
Location
PhysicalLocation (authority = marcorg); (displayLabel = Rutgers, The State University of New Jersey)
NjNbRU
Identifier (type = doi)
doi:10.7282/T3930R75
Genre (authority = ExL-Esploro)
ETD doctoral
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Rights

RightsDeclaration (ID = rulibRdec0006)
The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Yan
GivenName
Qing
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2013-09-16 19:02:16
AssociatedEntity
Name
Qing Yan
Role
Copyright holder
Affiliation
Rutgers University. Graduate School - New Brunswick
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License
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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Copyright protected
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Status
Open
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Permission or license
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ETD
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