Abstract
Heterotrimeric G proteins, consisting of alpha, beta, and gamma subunits, are implicated in major signal transduction pathways controlling a diversity of functions in eukaryotic organisms. In the filamentous fungus Neurospora crassa, G proteins are implicated in the regulation of several environmental responses. As a first step in studying the role of G proteins in these processes, we have cloned the genes for two alpha subunits, gna-1 and gna-2, from Neurospora. The genes are located on different chromosomes and are differentially regulated during asexual development. The encoded proteins (Gna-1 and Gna-2) are the same size as members of the Gi-alpha family (approximately 40 kDa). The Gna-1 protein sequence is 55% identical overall to members of the Gi family and contains the consensus sequences for ADP-ribosylation by pertussis toxin and incorporation of myristic acid, which are found in this group. These properties make Gna-1 the first identified microbial alpha subunit to be a member of any class. Furthermore, incubation of a N. crassa plasma membrane fraction with pertussis toxin results in ADP-ribosylation of a protein substrate which is the approximate size of Gna-1. The predicted Gna-2 protein sequence does not share a high degree of sequence identity with the Gi class. However, the coding region contains at least one intron in a position conserved in the Gi family. We propose that the Gi family of alpha subunits is ancient and during evolution may have first appeared in filamentous fungi.
Highlights
Heterotrimeric G proteins, consisting of a, j3, and y G-a protein in thepsreocesses in higher organisms isdifficult
The Gna-1 protein sequence is 55%identical overall to members of the Gi family and contains the consensus sequences for ADP-ribosylation by pertussis toxin and incorporation of myristic acid, which are found in this group
The three mammalianmembers of the Gi-a subfamily are variously implicated in inhibition of adenylylcyclase activity [6], regulation of ion channels,andcontrol of vesicular traffic throughthetrans-Golginetwork[4].Both rod and cone transducin are coupled to rhodopsin photoreceptors and activate retinal cGMP phosphodiesterase during tvhiesual cascade [8]
Summary
N. crassa genomic DNA was digested with the indicated restriction enzymes and subjected to Southern analysis as described under "Experimental Procedures." Duplicate blots were probed with either the gnu-1 or gnu-2 PCR products. The phage were amplified and thecDNA inserts excised using R408 helper phage as described elsewhere (Stratagene) In this way a -950-bp EcoRI-EcoRV fragment corresponding to the incomplete cDNA for gnu-1 was obtained. The carboxyl terminus of gnu-1 was obtained by screening a N. crassa genomic DNA cosmid library (pMOcosX [24]). Specific oligonucleotides corresponding to the region of gnu-1 amplified by the OMP19 and Ta29primers were synthesized These oligonucleotides during sexual development [16],and fertilized female struc- were used in initial PCRs with pooled DNA representing all cosmid turesexhibit positive phototropism to bluelight [17]. RFLP, restriction fragment-linked polymorphism; bp, base pair; kb, (gnu-1) andBstXI (gnu-2) yielded restriction fragment-linked polykilobase pair
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
