Analysis of yeast proteins with immunological or sequence similarities to mammalian PKC isozymes

When this project began, its principal aims were the isolation and characterisation of additional members of the yeast PKC family, which were believed to exist on the basis of biochemical data obtained in our laboratory, and others worldwide. One such gene, PKC2, was identified in our laboratory (Simon et al., 93) and a thorough functional analysis of its respective peptide product, Pkc2p was to have formed a major part of this project. In November 1994, a paper was published which cast serious doubts on the authenticity of the PKC2 gene (Levin et al., 94). In order for this study to continue, it was necessary to verify the existence of PCK2 . Nucleotide sequencing of all the recombinants purportedly used to generate the PKC2 open reading frame failed to reveal any of the hallmark sequences which allowed its categorisation as a member of the PKC family.;Nevertheless, the weight of biochemical evidence suggested that other PKC-like proteins did exist in yeast. In an attempt to identify the protein(s) responsible for these activities, an immunological screen was performed using polyclonal antisera known to recognise mammalian PKC isoforms. This screening failed to detect any such proteins but cross-reactivity was observed with the product of the YOR080w open reading frame. A thorough functional analysis of the YOR080w gene product, implicates the protein in the normal progression of the yeast cell cycle, with the defect manifesting itself at the spindle assembly checkpoint.