Genetic relationships between the RACK1 homolog cpc-2 and heterotrimeric G protein subunit genes in Neurospora crassa

Abstract

Receptor for Activated C Kinase-1 (RACK1) is a multifunctional eukaryotic scaffolding protein with a seven WD repeat structure. Among their many cellular roles, RACK1 homologs have been shown to serve as alternative Gβ subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gβ subunit gnb-1 and other G protein signaling components in the multicellular filamentous fungus Neurospora crassa. Results from cell fractionation studies and from fluorescent microscopy of a strain expressing a CPC-2-GFP fusion protein revealed that CPC-2 is a cytoplasmic protein. Genetic epistasis experiments between cpc-2, the three Gα genes (gna-1, gna-2 and gna-3) and gnb-1 demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height, while deletion of cpc-2 mitigates the increased macroconidiation on solid medium observed in Δgnb-1 mutants. Δcpc-2 mutants inappropriately produce conidiophores during growth in submerged culture and mutational activation of gna-3 alleviates this defect. Δcpc-2 mutants are female-sterile and fertility could not be restored by mutational activation of any of the three Gα genes. With the exception of macroconidiation on solid medium, double mutants lacking cpc-2 and gnb-1 exhibited more severe defects for all phenotypic traits, supporting a largely synergistic relationship between GNB-1 and CPC-2 in N. crassa.

A WD40 Protein Encoding Gene Fvcpc2 Positively Regulates Mushroom Development and Yield in Flammulina velutipes

“…Meanwhile, another strain (cpc-2; cpc-2), in which N. crassa cpc-2 was transformed into the cpc-2 mutant and used as a control strain. Similar to the previous report (Garud et al, 2019), the cpc-2 strain grew slower and produced less conidia than N. crassa wild type. Both the Fvcpc2; cpc-2 strain and the cpc-2; cpc-2 strain displayed the wild-type colony growth rate and wild-type conidial production ability (Figure 3), indicating Fvcpc2 can functionally replace the role of N. crassa CPC-2 in vegetative growth and asexual sporulation.…”

“…Therefore, the regulation of cAMP production is likely to be an important regulatory mechanism of FvCPC2 in morphological development. CPC-2 functions as the downstream of GNA-2 and the upstream of GNB-1 in hyphal growth and perithecial development in N. crassa, respectively (Garud et al, 2019). Since FvCPC2 has the similar role as CPC-2 of N. crassa in vegetative growth and fruiting body development, FvCPC2 might interact with the G protein signaling as CPC-2 does in F. velutipes.…”

Heterotrimeric G-Protein Signaling Is Required for Cellulose Degradation in Neurospora crassa

“…To investigate whether this is also the case for VM-Avicel, we isolated membrane fractions from VM-Avicel-grown cell pads and used Western blotting to determine levels of the different G protein subunits. Since CPC-2 is cytoplasmic in N. crassa ( 25 ), whole-cell extracts were used to determine protein levels in the various strains.…”

“… N. crassa uses G proteins to regulate basal hyphal growth, sexual development, asexual sporulation, and stress responses ( 13 , 18 – 23 ). N. crassa possesses three Gα proteins (GNA-1, GNA-2, and GNA-3) ( 24 ), two Gβ proteins (GNB-1 and CPC-2) ( 25 ), and a Gγ that associates with GNB-1 (GNG-1) ( 26 ). N. crassa also has two protein kinase A (PKA) catalytic subunits ( 27 , 28 ) that are predicted downstream targets of cAMP produced by the adenylyl cyclase CR-1 ( 29 ).…”

“…In this study, we embarked on a systematic analysis of cellulose phenotypes for all known G protein subunits and adenylyl cyclase. For this work, we utilized deletion mutants for all six G protein subunits ( 24 , 25 , 39 ) and strains expressing constitutively activated GTPase-deficient versions of each Gα subunit gene ( 40 ) ( gna-1 Q204L , gna-2 Q205L , and gna-3 Q208L ) (see Table S1 in the supplemental material). Our results provide evidence for transcriptional, posttranscriptional, and cAMP-dependent and -independent control of cellulase enzymes via G-protein signaling in N. crassa .…”