Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

Abstract

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.

A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

“…This number is consistent with previous estimates of 0.2-2% sequence divergence in coding regions between Oak Ridge and Mauriceville (Dillon and Stadler 1994;Nelson et al 1997;Coulter and Marzluf 1998). Potential mismatches were assessed using the NQS algorithm (Altshuler et al 2000), which takes into account the sequencing quality of both the mismatched and neighboring bases, and 4338 high-confidence putative SNPs (pSNPs) were retained ( Figure 1A, supplemental online database http:/ /www.broad.mit.edu/annotation/genome/ neurospora/).…”

A highly specific pathogen‐responsive promoter element from the immediate‐early activated CMPG1 gene in Petroselinum crispum

“…It is tempting to speculate that the CMPG domain, showing conspicuous similarity to the RING‐finger domain, constitutes a novel type of structurally and functionally important domain possibly mediating DNA or protein interactions. The additionally occurring serine/threonine‐rich domain may then be involved in transactivation, similarly as described for various other cases (Coulter and Marzluf, 1998; Rothermel et al ., 1997; Serra et al ., 1998; Tanaka and Herr, 1990).…”

Localization and light‐dependent phosphorylation of white collar 1 and 2, the two central components of blue light signaling in Neurospora crassa

“…In order to show the purity of the nuclear fractions, two control proteins were used as markers. CYS‐3 is a positive‐acting regulatory protein involved in the control of sulfur metabolism and is localized in the nucleus [25,26]. In contrast, the Neurospora nitrate reductase, which is only present in nitrate‐induced, de‐repressed Neurospora mycelia, is generally considered to be a soluble, cytoplasmic enzyme and has also been found to be associated with the plasmalemma and tonoplast membranes in vivo [27,28].…”