E-Syts, a family of membranous Ca2+-sensor proteins with multiple C2 domains.
C(2) domains are autonomously folded protein modules that generally act as Ca(2+)- and phospholipid-binding domains and/or as protein-protein interaction domains. We now report the primary structures and biochemical properties of a family of evolutionarily conserved mammalian proteins, referred to as E-Syts, for extended synaptotagmin-like proteins. E-Syts contain an N-terminal transmembrane region, a central juxtamembranous domain that is conserved from yeast to human, and five (E-Syt1) or three (E-Syt2 and E-Syt3) C-terminal C(2) domains. Only the first E-Syt C(2) domain, the C(2)A domain, includes the complete sequence motif that is required for Ca(2+) binding in C(2) domains. Recombinant protein fragments of E-Syt2 that include the first C(2) domain are capable of Ca(2+)-dependent phospholipid binding at micromolar concentrations of free Ca(2+), suggesting that E-Syts bind Ca(2+) through their first C(2) domain in a phospholipid complex. E-Syts are ubiquitously expressed, but enriched in brain. Expression of myc-tagged E-Syt proteins in transfected cells demonstrated localization to intracellular membranes for E-Syt1 and to plasma membranes for E-Syt2 and E-Syt3. Structure/function studies showed that the plasma-membrane localization of E-Syt2 and E-Syt3 was directed by their C-terminal C(2)C domains. This result reveals an unexpected mechanism by which the C(2)C domains of E-Syt2 and E-Syt3 functions as a targeting motif that localizes these proteins into the plasma membrane independent of their transmembrane region. Viewed together, our findings suggest that E-Syts function as Ca(2+)-regulated intrinsic membrane proteins with multiple C(2) domains, expanding the repertoire of such proteins to a fourth class beyond synaptotagmins, ferlins, and MCTPs (multiple C(2) domain and transmembrane region proteins).
Min
Sang-Won
SW
Department of Neuroscience and Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111, USA.
Chang
Wen-Pin
WP
Südhof
Thomas C
TC
D020816:Amino Acid Motifs; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005089:Exocytosis; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D010743:Phospholipids / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D050857:Synaptotagmins / Q000737:chemistry / Q000502:physiology*
D010743:Phospholipids; D011994:Recombinant Proteins; D050857:Synaptotagmins; D002118:Calcium