Purification and characterization of Snf1 kinase complexes containing a defined Beta subunit composition.
The Snf1 kinase complex of Saccharomyces cerevisiae contains one of three possible beta subunits encoded by either SIP1, SIP2, or GAL83. Snf1 kinase complexes were purified from cells expressing only one of the three beta subunits using a tandem affinity purification tag on the C terminus of the Snf1 protein. The purified kinase complexes were enzymatically active as judged by their ability to phosphorylate a recombinant protein containing the Snf1-responsive domain of the Mig1 protein. The Snf1 kinase complexes containing Gal83 or Sip2 as the beta subunit showed comparable and high levels of activity, whereas the Sip1-containing enzyme was significantly less active. Examination of the protein composition of the purified Snf1 enzyme complexes indicated that the Sip1 protein was present in substoichiometric levels. Increased gene dosage of SIP1 rescued the ethanol growth defect observed in cells expressing Sip1 as their only beta subunit and increased the in vitro activity of Snf1 kinase purified from these cells. Our studies indicate that the reduced activity of Snf1-Snf4-Sip1 kinase is due to low level of Sip1 accumulation rather than a limited ability of the Sip1 form of the enzyme to direct phosphorylation of specific substrates.
Nath
Nandita
N
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
McCartney
Rhonda R
RR
Schmidt
Martin C
MC
D005838:Genotype; D007700:Kinetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000302:isolation & purification; D021122:Protein Subunits / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D021122:Protein Subunits; D011994:Recombinant Proteins; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases