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Identification and characterization of APP1-encoded phosphatidate phosphatase in Saccharomyces cerevisiae

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TitleInfo
Title
Identification and characterization of APP1-encoded phosphatidate phosphatase in Saccharomyces cerevisiae
Name (type = personal)
NamePart (type = family)
Chae
NamePart (type = given)
Minjung
NamePart (type = date)
1981-
DisplayForm
minjung chae
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Carman
NamePart (type = given)
George M.
DisplayForm
George M. Carman
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
chair
Name (type = personal)
NamePart (type = family)
Han
NamePart (type = given)
Gil-Soo
DisplayForm
Gil-Soo Han
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Quardro
NamePart (type = given)
Loredana
DisplayForm
Loredana Quardro
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Chikindas
NamePart (type = given)
Michael
DisplayForm
Michael Chikindas
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Dixon
NamePart (type = given)
Joseph L.
DisplayForm
Joseph L. Dixon
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)
2013-01
Place
PlaceTerm (type = code)
xx
Language
LanguageTerm (authority = ISO639-2b); (type = code)
eng
Abstract (type = abstract)
Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP activity is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1Δ dpp1Δ lpp1Δ triple mutant indicated another gene(s) encoding the enzyme. I purified PAP activity from the pah1Δ dpp1Δ lpp1Δ triple mutant by salt-extraction of mitochondria, followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, I found that APP1, a gene whose molecular function has been unknown, confers ~30 % PAP activity of wild type cells. The overexpression of APP1 in the pah1Δ dpp1Δ lpp1Δ mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p that was heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1Δ mutation into the pah1Δ dpp1Δ lpp1Δ mutant resulted in a complete loss of PAP activity, indicating that the enzyme in S. cerevisiae is encoded by APP1, PAH1, DPP1, and LPP1. Protein A-tagged App1p PAP was expressed and purified from S. cerevisiae. The App1p PAP activity followed saturation kinetics with respect to the molar concentration of phosphatidic acid (PA) (Km = 0.5 mM) but followed positive cooperative (Hill number of ~2.3) kinetics with respect to the surface concentration of PA (Km = 2.0 mol %) in Triton X-100/PA mixed micelles. The App1p also exhibited lysoPA phosphatase and diacylglycerol pyrophosphate phosphatase activities. The order of substrate preference was phosphatidic acid> diacylglycerol pyrophosphate > lysoPA. The maximum PAP activity was dependent on Mg2+ ions at pH 7.5 at 30 ⁰C. The activation energy for the reaction was 16.5 kcal/mol, and the enzyme was labile above 40 ⁰C. The App1p PAP activity was inhibited by Ca2+, Mn2+, Zn2+, N-ethylmaleimide, propranolol, nucleotides, diacylglycerol, phosphatidylethanolamine, phosphatidylcholine, and sphinganine, while lysoPA, cardiolipin, phosphatidylglycerol, phosphatidylserine, sphingosine and sphingosine 1-phosphate stimulated the activity. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity.
Subject (authority = RUETD)
Topic
Food Science
Subject (authority = ETD-LCSH)
Topic
Phosphatidate phosphatase
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_4390
PhysicalDescription
Form (authority = gmd)
electronic resource
InternetMediaType
application/pdf
InternetMediaType
text/xml
Extent
xi, 124 p. : ill.
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
Note (type = vita)
Includes vita
Note (type = statement of responsibility)
by Minjung Chae
Subject (authority = ETD-LCSH)
Topic
Saccharomyces cerevisiae
Identifier (type = hdl)
http://hdl.rutgers.edu/1782.1/rucore10001600001.ETD.000067639
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/T3VM4B0Q
Genre (authority = ExL-Esploro)
ETD doctoral
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RightsDeclaration (ID = rulibRdec0006)
The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
chae
GivenName
minjung
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2012-11-08 14:35:56
AssociatedEntity
Name
minjung chae
Role
Copyright holder
Affiliation
Rutgers University. Graduate School - New Brunswick
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Name
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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Open
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