Identification and characterization of the components of the ER calcium network have provided important information to how these components may be regulated individually. A tight coordination of these components is, however, necessary to regulate both calcium fluxes, i.e. the amplitude and frequency of the cytosolic signal, as well as the internal ER calcium level. The challenge therefore lies in how these components act together, and affect each other, to achieve this regulation. While several aspects of how such coordination affects the calcium status of the ER/SR are becoming resolved in animal cells, analogous information is still largely lacking in plants.
The basic regulatory function is achieved through transcriptional coordination of the various components. This may be facilitated by the level of luminal ER calcium level, which works as a transcriptional switch for several components of the ER calcium network in animal cells (Waser et al. 1997). Both SERCA3 and Crt expressions are induced in response to depletion of ER calcium levels, e.g. by treatments with the ER Ca2+-pump inhibitor thapsi-gargin or the calcium ionophore A23187, implying an attempt of the cell to increase the Ca2+-holding potential of the ER (Waser et al. 1997; Liu et al. 2002).
The ER luminal proteins Cnx and Crt may coordinate the ER calcium dynamics in animal cells, i.e. influx, efflux and holding potential (Camacho and Lechleiter 1995; Michalak et al. 1999; Arnaudeau et al. 2002). Cnx and Crt can modulate the uptake of calcium by direct interactions with the Ca2+-pump SERCA2B (Camacho and Lechleiter 1995; Roderick et al. 2000). In contrast to other SERCAs which consist of ten transmembrane segments, SERCA2B has eleven transmembrane regions (Bayle et al. 1995). Consequently, the carboxy terminus of the protein is located within the ER lumen. Crt may directly interact with the luminal carboxy terminus of SERCA2B, a characteristic possibly regulated by the glycosylation status of the pump (John et al. 1998). Coex-pression of Crt and SERCA2B in Xenopus oocytes revealed that Crt effectively inhibits IP3-mediated calcium oscillations, resulting in a sustained elevation of cytosolic calcium (John et al. 1998). In addition, Cnx may also affect SERCA2B activity, possibly through a direct interaction with the pump (Roderick et al. 2000). Analogous to Crt, coexpression of Cnx and SERCA2B in Xenopus oocytes caused sustained elevation of cytosolic calcium, generated through inhibition of SERCA2B. The inhibition was promoted by phosphorylation of a serine residue in the cytosolic carboxy-terminal of Cnx (Roderick et al. 2000). The two chaperones Cnx and Crt may thus both affect the ER
Ca2+-uptake, coordinated through either internal ER processes or through cytosolic phosphorylation events.
Plants lack a SERCA2B homolog with a C-terminal luminal extension, and a direct effect on Ca2+-fluxes by any luminal ER Ca2+-binding proteins remains to be demonstrated. However, the activity of the Ca2+-pump ACA2 may be regulated by phosphorylation (Hwang et al. 2000). The phosphorylation occurs in the cytosolic N-terminal region and inhibited the activity of the pump (Fig. 5). As described above, CaM may activate ACA2 by interacting with the autoinhibitory N-terminal domain of the pump. When CaM was bound to the pump, the inhibitory phosphorylation was prevented (Hwang et al. 2000). The "signalling" dynamics of the cytosol may thus orchestrate an activation or inhibition of ACA2. The phosphorylation may be mediated through a CDPK referred to as CPK1 (Hwang et al. 2000). So far only one CDPK, CDPK2, has been shown to localize to the ER (Lu and Hrabak 2002). Whether this CDPK phosphorylates ACA2 in vivo has not yet been tested.
Crt may also be post-translationally modified through phosphorylation and glycosylation (Baldan et al. 1996; Li et al. 2003; Persson et al. 2003). In vitro studies suggest that the phosphorylation is mediated through a casein kinase II/CK2 (Baldan et al. 1996). Whether this kinase can enter the ER lumen remains undetermined. Although the glycosylation status of animal Crts is suggested to mediate a cellular redistribution of the protein, no physiological functions for either glycosylation nor phosphorylation are so far apparent for plant Crts.
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