RESUMO
A DNA sequence homologous to a G alpha s DNA probe, and the corresponding G alpha s protein (stimulatory alpha-subunit of GTP-binding protein) were detected in Phycomyces blakesleeanus. The protein was demonstrated in membrane fractions of dormant spores of this fungus using three different experimental approaches. Photoaffinity-labelling experiments with [alpha-32P]GTP of the membrane fraction revealed two bands, of 56 and 32 kDa. The 56 kDa GTP-binding protein was detected by this method in all the stages of early development and growth investigated. Also, a spore protein of 56 kDa was ADP-ribosylated by cholera toxin, and a 56 kDa protein was detected by Western blotting with a specific antibody against mammalian G alpha s. These results indicate that G alpha s (56 kDa) is present in dormant spores of P. blakesleeanus. Using the ADP-ribosylation and Western blotting assays, G alpha s was detected during all stages of spore germination before the hyphae became highly branched, but it was not detected in the branched hyphae that formed 18 h after the initiation of spore germination. Therefore, G alpha s is expressed differentially during Phycomyces development.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Phycomyces/fisiologia , Adenosina Difosfato Ribose/metabolismo , Marcadores de Afinidade , Toxina da Cólera/farmacologia , DNA Fúngico/genética , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Guanosina Trifosfato , Phycomyces/genética , Phycomyces/crescimento & desenvolvimento , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologiaRESUMO
The presence of protein kinase C (PKC), a key enzyme in signal transduction, has not been investigated in fungal cells. The phorbol ester TPA, an activator of PKC, may be used as an indicator of the presence and role of PKC in Phycomyces blakesleeanus spores. Activation of spore germination by acetate was prevented by 6 nM TPA. The TPA analog 4 alpha PDD, an ineffective activator of PKC, did not affect spore germination. 3 mM dbcAMP, on the other hand, reversed the inhibition of germination caused by TPA. TPA-stimulated protein kinase activity was detected in spores. The possible relationship between PKC and the increased levels of cAMP that accompany the induction of spore germination is discussed.