SummaryA cAMP-activatable Ca 2þ -dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa. Using a PCR approach, A. nidulans and N. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans, it is required to achieve wild-type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post-translational regulatory mechanisms, in particular cAMP-dependent protein kinase-mediated phosphorylation.

The first step of the hexosamine pathway is the formation of glucosamine-6-phosphate from fructose-6-phosphate and L-glutamine, a reaction catalyzed by L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase) (14). In B. emersonii amidotransferase is a dimer of two apparently identical 76-kDa subunits. This enzyme exists in two forms, which are interconvertible by phosphorylation or dephosphorylation of the serine residue(s) (12,25). Uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), the end product of hexosamine synthesis and also the substrate for chitin biosynthesis, specifically inhibits the activity of the phosphorylated form of the enzyme (12). In zoospores, the estimated concentration of UDP-GlcNAc (about 400 ,uM) is sufficient to completely inhibit the phosphorylated form of the amidotransferase (Ki for UDP-GlcNAc = 5 ,uM). Early during encystment, dephosphorylation of the enzyme allows it to escape inhibition by 25 (12,25). Although previous observations indicate that the dephosphorylation of amidotransferase is stimulated by magnesium (2, 12, 13), very little is known about the protein phosphatases (PPases) implicated in this process.The purpose of the present study was to characterize the PPases during the life cycle of B. emersondi and to identify those involved in the developmentally regulated control of hexosamine synthesis. To approach this problem we have used an improved procedure described by Cohen and coworkers for identifying and quantifying the serine-threonine PPase types 1, 2A, and 2C (PP1, PP2A, and PP2C) in animal tissues, yeast cells, plants, and protozoans (8, 9, 20, 22). Here we show that these three types of PPases are present in B. emersonii extracts and that amidotransferase is dephosphorylated by both PP2A and PP2C.

Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid-and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.