Interactions between conserved domains within homodimers in the BIG1, BIG2, and GBF1 Arf guanine nucleotide exchange factors.
Guanine nucleotide exchange factors carrying a Sec7 domain (ArfGEFs) activate the small GTP-binding protein Arf, a major regulator of membrane remodeling and protein trafficking in eukaryotic cells. Only two of the seven subfamilies of ArfGEFs (GBF and BIG) are found in all eukaryotes. In addition to the Sec7 domain, which catalyzes GDP/GTP exchange on Arf, the GBF and BIG ArfGEFs have five common homology domains. Very little is known about the functions of these noncatalytic domains, but it is likely that they serve to integrate upstream signals that define the conditions of Arf activation. Here we describe interactions between two conserved domains upstream of the Sec7 domain (DCB and HUS) that determine the architecture of the N-terminal regions of the GBF and BIG ArfGEFs using a combination of biochemical, yeast two-hybrid, and cellular assays. Our data demonstrate a strong interaction between DCB domains within GBF1, BIG1, and BIG2 to maintain homodimers and an interaction between DCB and HUS domains within each homodimer. The DCB/HUS interaction is mediated by the HUS box, the most conserved motif in large ArfGEFs after the Sec7 domain. In support of the in vitro data, we show that both the DCB and the HUS domains are necessary for GBF1 dimerization in mammalian cells and that the DCB domain is essential for yeast viability. We propose that the dimeric DCB-HUS structural unit exists in all members of the GBF and BIG ArfGEF groups and in the related Mon2p family and probably serves an important regulatory role in Arf activation.
Ramaen
Odile
O
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Joubert
Alexandra
A
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Simister
Philip
P
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Belgareh-Touzé
Naïma
N
Institut Jacques Monod-CNRS, Universités Paris VI et VII, 75251 Paris, France.
Olivares-Sanchez
Maria Conception
MC
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Zeeh
Jean-Christophe
JC
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Chantalat
Sophie
S
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Golinelli-Cohen
Marie-Pierre
MP
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Jackson
Catherine L
CL
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Biou
Valérie
V
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and.
Cherfils
Jacqueline
J
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France and. Electronic address: cherfils@lebs.cnrs-gif.fr.
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019281:Dimerization; D004789:Enzyme Activation / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D020798:Two-Hybrid System Techniques
C119087:ARFGEF1 protein, human; C120445:GBF1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D020727:ADP-Ribosylation Factors