Yeast chitin synthase 2 activity is modulated by proteolysis and phosphorylation.
Saccharomyces cerevisiae Chs2 (chitin synthase 2) synthesizes the primary septum after mitosis is completed. It is essential for proper cell separation and is expected to be highly regulated. We have expressed Chs2 and a mutant lacking the N-terminal region in Pichia pastoris in an active form at high levels. Both constructs show a pH and cation dependence similar to the wild-type enzyme, as well as increased activity after trypsin treatment. Using further biochemical analysis, we have identified two mechanisms of chitin synthase regulation. First, it is hyperactivated by a soluble yeast protease. This protease is expressed during exponential growth phase, when budding cells require Chs2 activity. Secondly, LC-MS/MS (liquid chromatography tandem MS) experiments on purified Chs2 identify 12 phosphorylation sites, all in the N-terminal domain. Four of them show the perfect sequence motif for phosphorylation by the cyclin-dependent kinase Cdk1. As we also show that phosphorylation of the N-terminal domain is important for Chs2 stability, these sites might play an important role in the cell cycle-dependent degradation of the enzyme, and thus in cell division.
Martínez-Rucobo
Fuensanta W
FW
Max Planck Institute of Biophysics, Department of Structural Biology, Max-von-Laue-Strasse 3, Frankfurt am Main, Germany.
Eckhardt-Strelau
Luise
L
Terwisscha van Scheltinga
Anke C
AC
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002687:Chitin Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D002687:Chitin Synthase; C400821:chitin synthase 2; D010447:Peptide Hydrolases