The brain exocyst complex interacts with RalA in a GTP-dependent manner: identification of a novel mammalian Sec3 gene and a second Sec15 gene.
Ral is a small GTPase involved in critical cellular signaling pathways. The two isoforms, RalA and RalB, are widely distributed in different tissues, with RalA being enriched in brain. The best characterized RalA signaling pathways involve RalBP1 and phospholipase D. To investigate RalA signaling in neuronal cells we searched for RalA-binding proteins in brain. We found at least eight proteins that bound RalA in a GTP-dependent manner. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified these as the components of the exocyst complex. The yeast exocyst is a regulator of polarized secretion, docking vesicles to regions of the plasma membrane involved in active exocytosis. We identified the human FLJ10893 protein as the mammalian homologue of the yeast exocyst protein Sec3p. The exocyst complex did not contain the previously identified exocyst component rSec15, but a new homologue of both yeast Sec15p and rSec15, called KIAA0919. Western blots confirmed that two rat exocyst proteins, rSec6 and rSec8, bound active RalA in nerve terminals, as did RalBP1. Phospholipase D bound RalA in a nucleotide-independent manner. This places the RalA signaling system in mammalian nerve terminals, where the exocyst may act as an effector for activated RalA in directing sites of exocytosis.
Brymora
A
A
Children's Medical Research Institute, 214 Hawkesbury Road, Westmead NSW 2145, Australia.
Valova
V A
VA
Larsen
M R
MR
Roufogalis
B D
BD
Robinson
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D005089:Exocytosis; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D033921:Vesicular Transport Proteins; D020725:ral GTP-Binding Proteins
C489222:EXOC6B protein, human; D020033:Protein Isoforms; D011994:Recombinant Proteins; C060862:SEC15 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C492198:RALA protein, human; C492197:Rala protein, rat; D020725:ral GTP-Binding Proteins