Synaptosomal-associated protein 25 plasma membrane anchor
Synaptosomal-associated protein 25, a protein synthesized on free ribosomes that is recruited to the inner surface of the plasma membrane after palmitoylation of cysteine residues. Fused to a reporter it directs the reporter to the cytoplasmic face of the plasma membrane.
Origin: Synaptosomal-associated protein 25 (SNAP25)
Characteristics
Encodes a 205-amino-acid protein (615 nucleotides, initiator methionine and stop codon excluded), beginning AEDADMRN and ending ATKMLGSG, with four cysteines in the stretch CCGLFICPC. SNAP-25 is synthesized on free ribosomes and recruited to the inner surface of the plasma membrane after post-translational palmitoylation of cysteine residues, which adds a lipid anchor. As the N-terminal partner of an aequorin fusion in A7r5 cells it gives peripheral staining that persists after digitonin permeabilization, unlike cytosolic aequorin.
Applications: Targets aequorin to the cytoplasmic rim beneath the plasma membrane. Fused upstream of HA1-tagged wild-type aequorin or the low-affinity Asp119 to Ala mutant and expressed in rat aortic smooth muscle A7r5 cells, it reports subplasmalemmal Ca2+ that is about 10-fold above bulk cytosol in unstimulated cells, with peaks of 8 to 10 micromolar after 100 micromolar Ca2+ readdition and above about 120 micromolar after 1 mM.
Limitations: Tested only in A7r5 cells, with aequorin fused to the C-terminus of SNAP-25. Aequorin reconstitution in 1 mM Ca2+ medium falls to 11% (wild type) and 21% (Asp119 to Ala) of the Ca2+-free value, versus 88% for cytosolic aequorin. Wild-type aequorin saturates at high Ca2+ and about 90% is consumed within 2 min. Unrelated to the SNAP-tag labeling tag.
Sequence
References
- Marsault et al. (1997). Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells. EMBO J - Marsault 1997 SNAP25 Aequorin