Studies on suc1 in xenopus laevis

Suc1 was originally identified in S.pombe as a sequence which suppressed certain mutations of cdc2. Homologues were subsequently identified in S.cerevisiae, humans and a variety of plants. To facilitate the biochemical analysis of suc1, antisera generated from S.pombe p13^suc1 were used to detect a suc1 homologue in Xenopus laevis. This immunological approach identified a putative suc1 homologue based on several criteria: (1) it is recognised by more than one polyclonal antiserum; (2) it is recognised exclusively by affinity-purified antiserum; (3) it is not recognised by antiserum pre-incubated with p13^suc1; (4) it co-fractionates with active cdks following high-speed centrifugation; and (5) it shares the property with p13^suc1 of being heat stable at 70^oC. The purification of this protein from a Xenopus oocyte extract followed a similar procedure to that of bacterially-expressed p13^suc1: batch elution from two ion-exchange matrices and heat treatment at 70^oC, followed by anion-exchange and gel-filtration chromatography.

This final purification step indicated that it may exist as a multimer. Analysis of the developmental profile of the protein revealed a gradual rise throughout oogenesis, and relatively constant levels throughout maturation and embryogenesis. It is also present in adult tissue, suggesting that it may have a role without p34^cdc2. Co-purification of the suc1-like protein, p34^cdc2 and mitotic histone kinase activity was also demonstrated using both gel-filtration and cation-exchange chromatography. The amount of the suc1-like protein in this fraction did not decrease when protein synthesis was inhibited, suggesting that it co-purifies with a sub-population of p34^cdc2. The levels of this sub-population did not change appreciably throughout the cell cycle. Finally, the addition of p13^suc1 to Xenopus egg extracts resulted in inhibition of the rate of p34^cdc2 activation, but not its deactivation. This inhibition was overcome by the inhibition of type 2A phosphatases.(DX179704)