Molecular cloning and expression of the regulatory (RGl) subunit of the glycogen-associated protein phosphatase

Pauline M. Tang, Jeffry A. Bondor, Kristine M. Swiderek, Anna De Paoli-Roach

Research output: Contribution to journalArticle

143 Citations (Scopus)

Abstract

DNA clones encoding the glycogen-binding (RGl) subunit of glycogen-associated protein phosphatase were isolated from rabbit skeletal muscle λgt11 cDNA libraries. Overlapping clones provided an open reading frame of 3327 nucleotides that predicts a polypeptide of 1109 amino acids with a molecular weight of 124,257. Northern hybridization of rabbit RNA identified a major mRNA transcript of 7.5 kilobases present in skeletal, diaphragm, and cardiac muscle, but not in brain, kidney, liver, and lung. Southern analysis of rabbit genomic DNA digested with various restriction endonucleases gave rise to a single hybridizing fragment, suggesting that a single gene is present. Expression of the complete RGl subunit coding sequence in Escherichia coli generated a protein of apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 160,000, similar to the size of the polypeptide detected by Western immunoblot in rabbit skeletal muscle extracts. The RGl subunit shares significant homology with the Saccharomyces cerevisiae GAC1 gene product which is involved in activation of glycogen synthase and glycogen accumulation. The homology with GAC1 substantiates the role of this enzyme in control of glycogen metabolism. Hydropathy analysis of the RGl subunit amino acid sequence revealed the presence of a hydrophobic region in the COOH terminus, suggesting a potential association with membrane. This result suggests that the same phosphatase regulatory component may be involved in targeting the enzyme both to membranes and to glycogen.

Original languageEnglish
Pages (from-to)15782-15789
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number24
StatePublished - 1991

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Cloning
Phosphoprotein Phosphatases
Molecular Cloning
Glycogen
Rabbits
Muscle
Skeletal Muscle
Clone Cells
Genes
Molecular Weight
Molecular weight
Membranes
Amino Acids
Glycogen Synthase
Peptides
DNA
DNA Restriction Enzymes
Enzymes
Diaphragms
Diaphragm

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular cloning and expression of the regulatory (RGl) subunit of the glycogen-associated protein phosphatase. / Tang, Pauline M.; Bondor, Jeffry A.; Swiderek, Kristine M.; De Paoli-Roach, Anna.

In: Journal of Biological Chemistry, Vol. 266, No. 24, 1991, p. 15782-15789.

Research output: Contribution to journalArticle

Tang, Pauline M. ; Bondor, Jeffry A. ; Swiderek, Kristine M. ; De Paoli-Roach, Anna. / Molecular cloning and expression of the regulatory (RGl) subunit of the glycogen-associated protein phosphatase. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 24. pp. 15782-15789.
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