Protein phosphatase 2C, encoded by ptc1+, is important in the heat shock response of Schizosaccharomyces pombe.

Protein phosphatase 2C (PP2C), an Mg(2+)-dependent enzyme that dephosphorylates serine and threonine residues, defines one of the three major families of structurally unrelated eukaryotic protein phosphatases. Members of the two other families of protein phosphatases are known to have important cellular roles, but very little is known about the biological functions of PP2C. In this report we describe a genetic investigation of a PP2C enzyme in the fission yeast Schizosaccharomyces pombe. We discovered ptc1+ (phosphatase two C) as a multicopy suppressor gene of swo1-26, a temperature-sensitive mutation of a gene encoding the heat shock protein hsp90. The ptc1+ gene product is a 40-kDa protein with approximately 24% identity to a rat PP2C protein. Purified Ptc1 has Mg(2+)-dependent casein phosphatase activity, confirming that it is a PP2C enzyme. A ptc1 deletion mutant is viable and has approximately normal levels of PP2C activity, observations consistent with the fact that ptc1+ is a member of a multigene family. Although a ptc1 deletion mutant is viable, it has a greatly reduced ability to survive brief exposure to elevated temperature. Moreover, ptc1+ mRNA levels increase 5- to 10-fold during heat shock. These data, demonstrating that Ptc1 activity is important for survival of heat shock, provide one of the first genetic clues as to the biological functions of PP2C.

Genetic analysis of the relationships between the amoebal extranuclear spindle-organizing centre and the plasmodial intranuclear spindle-organizing centre of Physarum during conjugation.

Physarum amoebae possess an extranuclear spindle-organizing centre (abbreviated SPOC), located in a typical centrosome with a pair of associated centrioles while plasmodia possess an intranuclear SPOC without centrioles. In order to ascertain whether, during conjugation, the plasmodial SPOC is derived from the amoebal one or is not related to it, we have constructed amoebal strains possessing two and three SPOCs and we have used as a genetic marker the frequency of polycentric metaphases in order to evaluate the number of SPOCs in the plasmodia. The results of both symmetrical crosses, i.e. between amoebae possessing the same number of SPOCs, and asymmetrical crosses, i.e. between amoebae possessing a different number of SPOCs, show that: (1) the number of SPOCs in plasmodia is dependent upon the number of SPOCs in either one of the two parental amoeba; (2) in no cross does the number of plasmodial SPOCs equal the sum of the parental amoebal SPOCs, but it corresponds to that of only one parent without any polarity of transmission in asymmetrical crosses. These results are consistent with the following model: (1) plasmodial SPOCs are derived from the amoebal ones; and (2) one set of parental SPOCs is lost, destroyed or inactivated in the zygote.

Polypeptides from the myxomycete Physarum polycephalum interacting in vitro with microtubules.

Microtubule-interacting proteins have been studied in the lower eukaryote Physarum polycephalum. We show for the first time 1) the presence in Physarum amoebal crude extracts of at least six polypeptides that bind specifically to amoebal microtubules, 2) the binding between these proteins and mammalian microtubules, 3) the heat stability of two of these polypeptides (125 and 235 kDa), 4) the functional properties of a fraction containing a heat-soluble 125 kDa polypeptide, and 5) the phosphorylation of the 125 kDa polypeptide during two distinct periods of the cell cycle in Physarum synchronous plasmodia, first at late S/early G2 phase and second at late G2/prophase.

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum.

The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. The mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cytoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.