Protein Phosphorylation in Archaea

Simple item page
dc.contributor.authorThurston, Barbaraen
dc.contributor.committeechairKennelly, Peter J.en
dc.contributor.committeechairWhite, Robert H.en
dc.contributor.committeememberAnderson, Bruce M.en
dc.contributor.committeememberBevan, David R.en
dc.contributor.committeememberDean, Dennis R.en
dc.contributor.committeememberTanko, James M.en
dc.contributor.departmentBiochemistry and Nutritionen
dc.date.accessioned2014-03-14T20:22:14Zen
dc.date.adate1997-03-10en
dc.date.available2014-03-14T20:22:14Zen
dc.date.issued1997-10-03en
dc.date.rdate1998-03-10en
dc.date.sdate1998-07-25en
dc.description.abstractProtein phosphorylation constitutes an important mechanism for cellular regulation in both Eucarya and Bacteria. All living organisms evolved from a common progenitor; this implies that protein phosphorylation as a means of regulation also exists in Archaea. Previously, in the sulfur-dependent archaeon Sulfolobus solfataricus a gene was cloned encoding a protein-serine/threonine phosphatase that was similar to eucaryal protein-serine/threonine phosphatases type 1, 2A, and 2B. To identify protein phosphatases in other archaeons, oligonucleotides encoding conserved regions of eucaryal protein-serine/threonine phosphatases were used in the polymerase chain reaction to amplify genomic DNA from the methanogenic archaeon Methanosarcina thermophila. From the PCR reaction a fragment of DNA was isolated that encoded a portion of a protein phosphatase. Using this DNA fragment as a probe, the entire phosphatase gene was isolated. The amino acid sequence of the phosphatase encoded by this gene displayed greater than 30% identity with eucaryal protein-serine/threonine phosphatase type 1. The gene encoding the Methanosarcina phosphatase was expressed in Escherichia coli. The expressed protein exhibited protein serine phosphatase activity that was sensitive to inhibitors of eucaryal phosphatases such as okadaic acid, microcystin, calyculin, and tautomycin. In order to identify potential endogenous substrates of archaeal protein-serine/threonine phosphatases and kinases, a study was initiated to characterize the most prominent phosphoproteins in S. solfataricus. Cell extracts were incubated with [γ-³²P] ATP, MgCl₂, and MnCl₂, and the proteins in the extracts were separated by SDS-PAGE. Autoradiography of the gels revealed four prominent phosphoproteins with apparent molecular masses of 35, 46, and 50 kDa. N-terminal sequence analysis and enzymatic assays of the 35 kDa phosphoprotein identified this phosphoprotein as the a-subunit of succinyl-CoA synthetase. N-terminal sequence analysis and enzymatic assays revealed that the 50 kDa phosphoprotein was a hexosephosphate mutase. Neither the 50 kDa nor the 35 kDa phosphoprotein appeared to be the target of protein kinases or phosphatases. Therefore, while protein-serine phosphatases exist in Archaea, the targets of these phosphatases have yet to be determined.en
dc.description.degreePh. D.en
dc.identifier.otheretd-58821132974710en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-58821132974710/en
dc.identifier.urihttp://hdl.handle.net/10919/30617en
dc.publisherVirginia Techen
dc.relation.haspartetdthesis*.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectsuccinyl-CoA synthetaseen
dc.subjectSulfolobus solfataricusen
dc.subjectprotein-serine/threonine phosphataseen
dc.subjecthexose phosphate mutaseen
dc.subjectMethanosarcina thermophilaen
dc.titleProtein Phosphorylation in Archaeaen
dc.typeDissertationen
thesis.degree.disciplineBiochemistry and Nutritionen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
etdthesis*.pdf
Size:
379.17 KB
Format:
Adobe Portable Document Format
Download

Collections

Doctoral Dissertations