Differential complementation of a Neurospora crassa Galpha(i) mutation using mammalian Galpha protein genes.

Heterotrimeric (alphabetagamma) G proteins interact with sensory receptors to transduce signals to downstream effectors in eukaryotes. We previously reported that GNA-1 from Neurospora crassa is a microbial member of the Galphai family found in higher organisms. Deletion of gna-1 leads to female sterility, slower growth rates on normal and hyperosmotic solid medium, and increased resistance to heat and oxidative stress. In this study we compare mammalian genes for proteins of the Galphai sub-family (Galphai, Galphao, Galphat and Galphaz), and Galphas (which is not a member of the Galphai family) with the N. crassa gna-1 gene with respect to their ability to complement deltagna-1 phenotypes. Northern analysis detected full-length transcripts of all these genes, except that for Galphai, in N. crassa transformants. Measurements of pertussis toxin-catalyzed ADP-ribosylation and Western analysis showed that the GNA-1, Galphaz, Galphao and Galphas proteins were present in the respective transformed strains. Strains in which the mammalian Galpha protein could be detected were subjected to phenotypic testing. During the vegetative cycle, none of the mammalian Galpha genes complemented the thermotolerance phenotype of deltagna-1. However, the three expressed mammalian Galpha genes achieved at least partial complementation of the defects in vegetative apical extension rate. cAMP levels did not correlate with restoration of vegetative growth rate by the mammalian genes. During the sexual cycle, Galphao was the only mammalian Galpha gene that rescued the defect in female fertility characteristic of deltagna-1 strains. Alignment of GNA-1, Galphaz, Galphao and Galphas protein sequences revealed correlations between the observed complementation pattern and the degree of identity to GNA-1 in various functional motifs. The finding that Galphac gave the best restoration of vegetative growth but could not restore normal female fertility implies that GNA-1 regulates different pathways that are important for vegetative and sexual growth in N. crassa.

A eukaryotic protein, NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactive pigment.

The nop-1 gene from Neurospora crassa is predicted to encode a seven-helix protein exhibiting conservation with the rhodopsins of the archaeon Halobacterium salinarum. In the work presented here we have expressed this gene heterologously in the yeast Pichia pastoris, obtaining a relatively high yield of 2.2 mg of NOP-1 protein/L of cell culture. The expressed protein is membrane-associated and forms with all-trans retinal a visible light-absorbing pigment with a 534 nm absorption maximum and approximately 100 nm half-bandwidth typical of retinylidene protein absorption spectra. Its lambda(max) indicates a protonated Schiff base linkage of the retinal. Laser flash kinetic spectroscopy demonstrates that the retinal-reconstituted pigment undergoes a photochemical reaction cycle with a near-UV-absorbing intermediate that is similar to the M intermediates produced by transient Schiff base deprotonation of the chromophore in the photocycles of bacteriorhodopsin and sensory rhodopsins I and II. The slow photocycle (seconds) and long-lived intermediates (M and O) are most similar to those of the phototaxis receptor sensory rhodopsin II. The results demonstrate a photochemically reactive member of the archaeal rhodopsin family in a eukaryotic cell.

The nop-1 gene of Neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins.

Opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had been identified in animals and the Archaea but not in fungi or other eukaryotic microorganisms. Here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus Neurospora crassa. The nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues, including the conserved lysine residue that forms a Schiff base linkage with retinal. Evolutionary analysis revealed relatedness not only between NOP-1 and archaeal opsins but also between NOP-1 and several fungal opsin-related proteins that lack the Schiff base lysine residue. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Extensive analysis of Deltanop-1 strains did not reveal obvious defects in light-regulated processes under normal laboratory conditions. However, results from Northern analysis support light and conidiation-based regulation of nop-1 gene expression, and NOP-1 protein heterologously expressed in Pichia pastoris is labeled by using all-trans [3H]retinal, suggesting that NOP-1 functions as a rhodopsin in N. crassa photobiology.