Positive and negative regulation of a SNARE protein by control of intracellular localization.
In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.
Nakanishi
Hideki
H
Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, SUNY Stony Brook, Stony Brook, New York 11794-5215, USA.
de los Santos
Pablo
P
Neiman
Aaron M
AM
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005690:Galactose / Q000378:metabolism; D005838:Genotype; D005947:Glucose / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008081:Liposomes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013696:Temperature; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D008081:Liposomes; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010455:Peptides; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D005947:Glucose; D005033:Ethylmaleimide; D005690:Galactose