Regulation of synaptophysin degradation by mammalian homologues of seven in absentia.
Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm. Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved. Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia. We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association. Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes. In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin. Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway. Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin.
Wheeler
Tiffany C
TC
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Chin
Lih-Shen
LS
Li
Yankun
Y
Roudabush
Francine L
FL
Li
Lian
L
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D016716:PC12 Cells; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016708:Synaptophysin / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins
D018076:DNA, Complementary; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016708:Synaptophysin; C106347:UBE2E2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D005982:Glutathione Transferase; D008025:Ligases