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 Ca(2+), hippocalcin rapidly translocated to the same perinuclear compartment as NCS-1. Another member of the family, neurocalcin delta, also translocated to this region after a rise in Ca(2+) concentration. Permeabilization of transfected cells using digitonin caused loss of hippocalcin and neurocalcin delta in the absence of calcium, but in the presence of 10 microm Ca(2+), 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 Ca(2+). 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 Ca(2+) concentration. Dual imaging of hippocalcin-EYFP and cytosolic Ca(2+) concentration in Fura Red-loaded cells demonstrated the kinetics of the Ca(2+)/myristoyl switch in living cells and showed that hippocalcin rapidly translocated with a half-time of approximately 12 s after a short lag period when Ca(2+) was elevated. These results demonstrate that closely related Ca(2+) sensor proteins use their myristoyl groups in distinct ways in vivo in a manner that will determine the time course of Ca(2+) signal transduction.
Highlights
Tration in addition to global Ca2ϩ changes has been increasingly highlighted, and these local Ca2ϩ signals are likely to contribute to the specificity of Ca2ϩ actions (2)
Studies on protein dynamics have been complemented by increasing information on the differential use of local versus global Ca2ϩ signals in the regulation of cell function (2)
We have examined the localization of three members of the neuronal calcium sensor (NCS) family of Ca2ϩ sensors and demonstrate that, under the same conditions in the same cellular context, their N-terminal myristoyl groups are used in quite different ways to determine their localization
Summary
Tration in addition to global Ca2ϩ changes has been increasingly highlighted, and these local Ca2ϩ signals are likely to contribute to the specificity of Ca2ϩ actions (2). There is a large conformational change that ejects the myristoyl group from its pocket (16) This exposed hydrophobic tail is free to interact with the nonpolar cell membranes or hydrophobic protein domains. Hippocalcin is expressed most highly in hippocampal neurons (19) and the closely related neurocalcin ␦ (VILIP-3) in cerebellar Purkinje cells (20, 21) Biochemical studies with these proteins suggest Ca2ϩ-dependent membrane interactions (17, 18), but this has not been examined within intact cells. The functions of these two proteins are unknown. We demonstrate different uses of N-terminal myristoylation, which would generate distinct spatio-temporal Ca2ϩ sensing by members of the NCS family and have assessed the kinetics of the Ca2ϩ/myristoyl switch-dependent membrane translocation in living cells
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