The Ncypt1 gene from Neurospora crassa is located on chromosome 2: molecular cloning and structural analysis

Abstract

Small GTP-binding proteins are encoded by ras-like genes and play a central role in cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the Ascomycete fungus Neurospora crassa with Zmypt genes from Zea mays we have isolated a member of the ypt gene family, Ncypt1. The gene resides on a 4 kb fragment of genomic DNA and contains four introns, which interrupt the coding sequence of a protein of 203 amino acid residues. The Ncytp1 gene was assigned to a single-copy gene encoding a transcript of 1.5 kb and a protein of 26,000 daltons. The gene maps on linkage group IIR between DB0001 and ccg-2 close to the Fsr-3 locus. Analysis of the nucleotide sequence and the deduced protein sequence revealed a striking homology to yeast, mouse and human genes encoding small GTP-binding proteins that are related to the ras supergene family. Homology was most significant to ypt1 from Schizosaccharomyces pombe, Mus musculus and Homo sapiens sharing 84.8%, 82.3%, and 82.3% identity, respectively. Common domains present in other small GTP-binding proteins were identified in the predicted sequence of the NCYPT1 protein, and the arrangement of peptide motifs sharing similarity with well characterized, small GTP-binding proteins suggests that the NCYPT1 protein is a GTPase. The C-terminal region extending from amino acid residues 175 to 199 shares only weak amino acid sequence similarity with other eukaryotic GTPases. Like other RAS proteins the NCYPT1 protein contains two conserved C-terminal cysteine residues, suggesting post-translational modification(s) by fatty acylation required for membrane anchoring. The high degree of homology between the NCYPT1 protein and eukaryotic YPT proteins suggests that NCYPT1 could be involved in the control of secretory processes.

Characterization of Secretory Genes ypt1 / yptA and nsf1 / nsfA from Two Filamentous Fungi: Induction of Secretory Pathway Genes of Trichoderma reesei under Secretion Stress Conditions

“…awamori encodes a putative protein of 201 amino acids, having 1 amino acid difference compared to the srgB gene cloned from A. niger by Punt et al (36) and sharing 94% identity with the Trichoderma YPTI protein. The yptA gene has four introns at positions identical to those for introns in the T. reesei and N. crassa (13) ypt1 genes (Fig. 1).…”

“…Folding factors, such as Bip and protein disulfide isomerase (PDI), have been characterized at the gene level for some filamentous fungi (e.g., see references 28, 38, and 48). Of the genes involved in protein trafficking, results have hitherto been published for the ypt1 genes of Neurospora crassa (13) and Phytophthora infestans (5) and for homologs of the yeast SAR1 gene from A. niger and T. reesei (49). Punt et al (36) have cloned several secretionrelated small GTPases from A. niger, and Dumas et al (8) have cloned a homolog of yeast SEC4 from Colletotrichum linde-muthianum.…”

A Tip-High Gradient of a Putative Plasma Membrane SNARE Approximates the Exocytotic Gradient in Hyphal Apices of the Fungus Neurospora crassa

“…While our data best fit the gradient of exocytosis predicted by observed rates of cell wall synthesis, other factors may regulate vesicle exocytosis in hyphal tips. One such factor may be rab family GTPases, which regulate SNARE activity (Pelham, 1999;Finger and Novick, 1998), function in a rate-limiting step prior to vesicle fusion (Geppert and Sudhof, 1998), and have homologues in N. crassa (Heintz et al, 1992;our unpublished observations). Our observations that the PM t-SNARE gradient profiles do not correlate with hyphal growth rates, yet the intracellular t-SNARE staining gradients do support the existence of rab-like regulation.…”

The Secretion Pathway in Filamentous Fungi: A Biotechnological View

“…Preliminary analysis of srgC disruption mutants showed an abnormal hyphal phenotype and temperature-sensitive growth (R. Montijn et al, unpublished). Other cloned fungal GTPases include the ypt1 homologues of N. crassa (Heintz et al, 1992) and T. reesei and A. niger var. awamori (Saloheimo et al, 2000).…”