The B regulatory subunit of protein phosphatase 2A is required for completion of macroconidiation and other developmental processes in Neurospora crassa

Abstract

rgb-1, encoding the tentative B regulatory subunit of the type 2A Ser/Thr phosphatase in Neurospora crassa, was isolated from cDNA and genomic libraries. Based on analysis of cDNA and genomic clones, rgb-1 is 3387 nucleotides in length, contains seven putative introns and encodes a 461-amino-acid polypeptide. Intron I, which is 5' to the presumed translation initiation codon, contains a uORF encoding 34 amino acids. Intron VI undergoes alternative splicing. Inactivation of rgb-1 by the repeat-induced point (RIP) mutation procedure produced progeny that grow slowly, have abnormal hyphal morphology, are female sterile and produce abundant amounts of arthroconidia. The rgb-1RIP strain does not produce major constriction chains or mature macroconidia. Minor constriction chains are formed, yet the growth process reverts to hyphal elongation. Microscopic and genetic analyses indicate that rgb-1 is a regulator of the budding subroutine of the macroconidiation process and that arthroconidiation, which shares common early and late events with macroconidiation, is induced as a default mechanism for asexual reproduction in this fungus.

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

“…Because the catalytic subunits of PP1 and PP2A (encoded for by the ppp-1 and pph-1 genes, respectively) are essential for cell survival in Neurospora and other eukaryotes (Yatzkan and Yarden 1995;Zeke et al 2003), we used repeat-induced point mutation (RIP; Cambareri et al 1989) to obtain partially functional To examine whether these mutations led to the decrease of the phosphatase activity of PP1, phosphatase assay (see supplemental materials) was performed using extracts prepared from cultures grown in LL, a condition that the clock is not running. The 32 P-labeled phosphorylase a is used as the substrate of the assay in the presence or absence of inhibitor-2, a specific protein inhibitor for PP1 (Krebs and Fischer 1962;Yatzkan et al 1998;Cohen 2002).…”

“…Because the catalytic subunits of PP1 and PP2A (encoded for by the ppp-1 and pph-1 genes, respectively) are essential for cell survival in Neurospora and other eukaryotes (Yatzkan and Yarden 1995;Zeke et al 2003), we used repeat-induced point mutation (RIP; Cambareri et al 1989) to obtain partially functional mutants of these two genes. RIP introduces random, but exclusively G-C to A-T point mutations during sexual cycle.…”

“…This dimeric core can form trimeric complexes with a third variable regulatory subunit (B subunit), which regulates the activity, specificity, and the localization of the holoenzyme (Virshup 2000). To study the role of one of the PP2A holoenzymes in the Neurospora circadian clock, we studied the circadian clock phenotype in a mutant (rgb-1 RIP ), in which one of the Neurospora PP2A B subunits (RGB-1) had been disrupted by RIP (Yatzkan and Yarden 1999). RGB-1 is a highly conserved B subunit of PP2A found in fungi, plants, insects, and mammals.…”

“…Because both strains contained wild-type clocks, for simplicity, they were both called wild-type strains. All other strains described in this study contain the bd mutation, except for the rgb-1 RIP strain, which was described previously (Yatzkan and Yarden 1999). The FGSC 987 strain (without the bd mutation) was used as the control strain in experiments described in Figures 4 and 5C, while the bd,a strain was used as the control strain in the rest of the study.…”

Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit

“…A report by Yatzkan & Yarden (1999) has suggested a link between PP2A regulatory subunit rgb-1 and macroconidiation in N. crassa: inactivation of rgb-1 leads to a failure in the formation of mature macroconidia. In contrast, we observed that macroconidiation was activated in PP179 (Fig.…”