A cAMP-binding ectoprotein in the yeast Saccharomyces cerevisiae

tides 10, 593-595

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

BACKGROUND: 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. 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. RESULTS: Based 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). CONCLUSION: Our 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

Identification of a Guanine Nucleotide Exchange Factor for Arf3, the Yeast Orthologue of Mammalian Arf6

Small G proteins of the Arf and Rab families are fundamental to the organisation and activity of intracellular membranes. One of the most well characterised of these G proteins is mammalian Arf6, a protein that participates in many cellular processes including endocytosis, actin remodelling and cell adhesion. Exchange of GDP for GTP on Arf6 is performed by a variety of guanine nucleotide exchange factors (GEFs), principally of the cytohesin (PSCD) and EFA6 (PSD) families. In this paper we describe the characterisation of a GEF for the yeast orthologue of Arf6, Arf3, which we have named Yel1 (yeast EFA6-like-1) using yeast genetics, fluorescence microscopy and in vitro nucleotide exchange assays. Yel1 appears structurally related to the EFA6 family of GEFs, having an N-terminal Sec7 domain and C-terminal PH and coiled-coil domains. We find that Yel1 is constitutively targeted to regions of polarised growth in yeast, where it co-localises with Arf3. Moreover the Sec7 domain of Yel1 is required for its membrane targeting and for that of Arf3. Finally we show that the isolated Yel1 Sec7 domain strongly stimulates nucleotide exchange activity specifically on Arf3 in vitro

Expression and secretion of insect immune peptides in yeast.

Cellular and molecular aspects of insect immunit