Title page for ETD etd-58821132974710


Type of Document Dissertation
Author Thurston, Barbara
Author's Email Address
URN etd-58821132974710
Title Protein Phosphorylation in Archaea
Degree PhD
Department Biochemistry and Nutrition
Advisory Committee
Advisor Name Title
Anderson, Bruce M.
Bevan, David R.
Dean, Dennis R.
Tanko, James M.
Kennelly, Peter J. Committee Chair
White, Robert H. Committee Chair
Keywords
  • succinyl-CoA synthetase
  • Sulfolobus solfataricus
  • protein-serine/threonine phosphatase
  • hexose phosphate mutase
  • Methanosarcina thermophila
Date of Defense 1997-10-03
Availability unrestricted
Abstract
Protein 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 [g-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.

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