Abstract
BackgroundSmall G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown.ResultsBased on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7).ConclusionOur analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.
Highlights
Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP
Domain 'hunting' in BFA-Inhibited Guanine nucleotide exchange factor (BIG) and Golgi-associated BFA-resistant guanine nucleotide exchange Factor (GBF) subfamilies was complicated by the facts that the Sec7 domain is their only domain that could be identified from known domain repertoires, and that their poorly characterized non-catalytic regions were not found outside these ArfGEF subfamilies
We based our search of candidate structural domains in BIGs and GBFs on the bioinformatics analysis of their own sequences, taking advantage of the growing number of sequences from fully annotated genomes from mammals, insects, plants, nematode, and fungi, to which we included our annotation of Sec7-containing proteins from the newly sequenced genome of Paramecium
Summary
Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. Guanine Nucleotide Exchange Factors (GEFs) are obligatory components of signaling cascades regulated by small GTP-binding proteins (called small G proteins hereafter). Their biochemical activity is to stimulate the dissociation of the tightly bound GDP nucleotide from the small G protein in response to cellular signals. Each small G protein family features its own ensemble of GEFs characterized by a conserved catalytic domain responsible for nucleotide exchange, which is generally combined with non-catalytic domains that define the spatio-temporal conditions of activation. Little is known about the functions of the other domains, which are likely to determine intracellular localization of ArfGEFs and their responsiveness to specific signals
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