Differential use of myristoyl groups on neuronal calcium sensor proteins as a determinant of spatio-temporal aspects of Ca2+ signal transduction

Abstract

The localizations of three members of the neuronal calcium sensor (NCS) family were studied in HeLa cells. Using hippocalcin-EYFP and NCS-1-ECFP, it was found that their localization differed dramatically in resting cells. NCS-1 had a distinct predominantly perinuclear localization (similar to trans-Golgi markers), whereas hippocalcin was present diffusely throughout the cell. Upon the elevation of intracellular Ca2+, hippocalcin rapidly translocated to the same perinuclear compartment as NCS-1. Another member of the family, neurocalcin δ, also translocated to this region after a rise in Ca2+ concentration. Permeabilization of transfected cells using digitonin caused loss of hippocalcin and neurocalcin δ in the absence of calcium, but in the presence of 10 μM Ca2+, both proteins translocated to and were retained in the perinuclear region. NCS-1 localization was unchanged in permeabilized cells regardless of calcium concentration. The localization of NCS-1 was unaffected by mutations in all functional EF hands, indicating that its localization was independent of Ca2+. A minimal myristoylation motif (hippocalcin-(1-14)) fused to EGFP resulted in similar perinuclear targeting, showing that localization of these proteins is because of the exposure of the myristoyl group. This was confirmed by mutation of the myristoyl motif of NCS-1 and hippocalcin that resulted in both proteins remaining cytosolic, even at elevated Ca2+ concentration. Dual imaging of hippocalcin-EYFP and cytosolic Ca2+ concentration in Fura Redloaded cells demonstrated the kinetics of the Ca2+/myristoyl switch in living cells and showed that hippocalcin rapidly translocated with a half-time of ∼12 s after a short lag period when Ca2+ was elevated. These results demonstrate that closely related Ca2+ sensor proteins use their myristoyl groups in distinct ways in vivo in a manner that will determine the time course of Ca2+ signal transduction.