Downloaded from http://cshperspectives.cshlp.org/ on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Organellar Calcium Buffers Daniel Prins and Marek Michalak Department of Biochemistry, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Correspondence: [email protected] Ca2þ is an important intracellular messenger affecting many diverse processes. In eukaryotic cells, Ca2þ storage is achieved within specific intracellular organelles, especially the endo- plasmic/sarcoplasmic reticulum, in which Ca2þ is buffered by specific proteins known as Ca2þ buffers. Ca2þ buffers are a diverse group of proteins, varying in their affinities and capacities for Ca2þ, but they typically also carry out other functions within the cell. The wide range of organelles containing Ca2þ and the evidence supporting cross-talk between these organelles suggest the existence of a dynamic network of organellar Ca2þ signaling, mediated by a variety of organellar Ca2þ buffers. INTRACELLULAR Ca2þ DYNAMICS that they also serve other roles within the cell. These roles include catalyzing the correct fold- a2þ serves as an intracellular messenger in ing of other cellular proteins, regulating Ca2þ various cellular processes, including muscle C release and retention, and communicating in- contraction, gene expression, and fertilization formation about Ca2þ levels within organelles (Berridge et al. 2003). To use Ca2þ, the cell to other proteins. requires a readily mobilizable source of Ca2þ, the majority of which is found within the lumen of the endoplasmic reticulum (ER) and/or sar- ENDOPLASMIC RETICULUM coplasmic reticulum (SR), but is also located in the Golgi apparatus, peroxisomes, mitochon- The ER is a multifunctional organelle within the dria, and endolysosomal compartments. It is eukaryotic cell that serves as the single largest not surprising that Ca2þ levels are of central im- Ca2þ store inside nonstriated muscle cells. The portance in dictating the function of proteins ER is also responsible for functions as diverse that reside in intracellular organelles. Although as protein synthesis and posttranslational mod- some Ca2þ exists as free ions within these com- ification, lipid and steroid metabolism, and partments, much of it is buffered by specific drug detoxification (Michalak and Opas 2009). proteins, simply known as Ca2þ buffers. How- Within the ER lumen, the total concentration ever, these proteins are diverse in terms of struc- of Ca2þ is approximately 1 mM, with free ture, oligomerization, affinity and capacity for Ca2þ in the range of approximately 200 mM Ca2þ, and physical basis for binding Ca2þ and the remainder buffered via ER resident pro- ions, one commonality across Ca2þ buffers is teins (Michalak and Opas 2009). Most ER Ca2þ Editors: Martin D. Bootman, Michael J. Berridge, James W. Putney, and H. Llewelyn Roderick Additional Perspectives on Calcium Signaling available at www.cshperspectives.org Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a004069 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004069 1 Downloaded from http://cshperspectives.cshlp.org/ on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press D. Prins and M. Michalak buffering proteins also serve as ER chaperones showed that the tip of the P-domain protrusion or folding enzymes, responsible for correctly accounts for the binding site of ERp57, an protein folding that are transiting through oxidoreductase-folding enzyme (Frickel et al. the ER. 2002). The C-domain of calreticulin is enriched in negatively charged amino acid residues respon- Calreticulin sible for its Ca2þ-buffering capabilities (Naka- Calreticulin, a 46-kDa ER luminal resident pro- mura et al. 2001b). It binds Ca2þ with high tein, is responsible for buffering up to 50% of capacity (25 mol of Ca2þ per mol of protein) 2þ ER Ca in nonmuscle cells (Nakamura et al. and low affinity (Kd ¼ 2 mM) (Nakamura et al. 2001a; Nakamura et al. 2001b). Structurally, 2001b). The conformation of this region is calreticulin consists of three distinct domains: highly dependent on variations in Ca2þ concen- N, which is the amino-terminal and implicated trations within a physiological range (Corbett (together with the P-domain) in chaperone et al. 2000). SAXS studies indicate that the C- function; P, which is central, proline-rich, and domain of calreticulin may be globular (Nor- a structural backbone; and C, which is the gaard Toft et al. 2008). Ca2þ binding stabilizes carboxy-terminal and critical for Ca2þ buffer- the C-domain into a more compact, a-helical ing. The N-domain of calnexin, which is ho- conformation; the Ca2þ concentration required mologous to calreticulin, is primarily b-sheet to induce this change, 400 mM, is well within and globular, with high homology to the struc- the range of concentrations to which calreticu- ture of plant lectins, suggesting a role in the lin would be exposed physiologically (Villamil binding of monoglucosylated substrates to cal- Giraldo et al. 2009). reticulin as part of its chaperone role (Schrag et al. 2001). Recent work using small angle Calreticulin Gain-of-Function X-ray scattering (SAXS) showed that the N- and Loss-of-Function domain of calreticulin itself is indeed globular and fits well onto modeled calnexin (Norgaard Understanding the roles calreticulin and Ca2þ Toft et al. 2008). The N-domain conformation play at cellular and organismal levels requires is dynamic and is stabilized by oligosaccharide accounting for both its role as a chaperone binding (Saito et al. 1999; Conte et al. 2007) and as an ER luminal Ca2þ buffer. Cell culture and the binding of Ca2þ at a high-affinity site and animal models of calreticulin deficiency (Corbett et al. 2000; Conte et al. 2007), though (loss-of-function) and overexpression (gain- this binding does not affect its affinity for oligo- of-function) have shown how tight control of saccharides (Conte et al. 2007). protein folding and Ca2þ homeostasis, as ex- The P-domain of calreticulin is proline- erted by calreticulin, are necessary for proper rich, which suggests that it may show confor- function and development. mational flexibility. Its sequence contains two In mice, calreticulin deficiency (loss-of- pairs of repeated amino acid sequences, 1 and function) is lethal at embryonic day 14.5 be- 2, in the order 111222 (Fliegel et al. 1989). The cause of impaired cardiogenesis, manifested P-domain adopts an extended conformation as abnormally thin ventricular walls and im- with antiparallel b-sheets between the repeated proper myofibrillogenesis (Mesaeli et al. 1999). amino acid sequences; the domain as a whole The insufficiency in this pathway can be traced protrudes out from the N- and C-domains to a lack of nuclear translocation of NF-AT3 (Ellgaard et al. 2001a; Ellgaard et al. 2001b). The (nuclear factor of activated T-cells), which is ac- extended protrusion requires a b-hairpin turn tivated by calcineurin, a Ca2þ-dependent phos- at amino acid residues 238 to 241; small angle phatase. crt2/2 cells showed no cytoplasmic X-ray scattering (SAXS) analyses indicate that Ca2þ spike in response to the agonist bradyki- this is in a spiral-like conformation (Norgaard nin, indicating that the IP3 pathway to stimu- Toft et al. 2008). TROSY-NMR experiments late release of Ca2þ from the ER was affected 2 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004069 Downloaded from http://cshperspectives.cshlp.org/ on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Calcium Buffers (Mesaeli et al. 1999). Embryonic lethality could implicated in cardiac development, suggesting be rescued via heterologous expression of a con- that calreticulin may play multiple roles in con- stitutively active mutant of calcineurin in the trolling downstream gene transcription (Hat- heart, demonstrating that calreticulin’s role in tori et al. 2007). cardiogenesis depends on its regulation of intra- cellular Ca2þ dynamics from the ER lumen Immunoglobulin Binding (Guo et al. 2002). Calreticulin may regulate the 2þ Protein BiP/GRP78 IP3RinaCa -dependent manner (Camacho and Lechleiter 1995; Naaby-Hansen et al. BiP (immunoglobulin binding protein), also 2001). Furthermore, in a cell culture model, car- known as GRP78, similarly to calreticulin, is diomyocytes derived from crt2/ – embryonic an ER luminal resident protein known to play stem cells showed lower rates of myofibrillar an important role in binding to unfolded pro- development (Li et al. 2002), thought to involve teins and assisting in the attainment of the cor- transcriptional pathways regulated by Ca2þ rect conformations. Its most prominent roles (Lynch et al. 2006). The critical role of Ca2þ sig- are as a regulator of the unfolded protein re- naling is underscored by the observation that sponse, a player in ER stress, and a crucial com- myofibrillar development could be rescued in ponent of the protein translocation machinery crt2/ – cells by transient ionomycin treatment (Dudek et al. 2009). BiP/GRP78 deficiency (Li et al. 2002). Taken together, these investiga- (loss-of-function) is extremely detrimental and tions show that the absence of calreticulin is lethal at embryonic day 3.5 in mice (Luo et al. severely affects Ca2þ-regulated pathways, via 2006). In addition to its protein binding func- both calreticulin’s Ca2þ buffering and its regula- tions, BiP/GRP78 serves as an important lumi- tion of protein folding. nal Ca2þ buffer, likely responsible for buffering Calreticulin overexpression (gain-of-func- approximately 25% of the total ER Ca2þ load tion) is also detrimental to the maintenance of (Lievremont et al. 1997). As BiP/GRP78 is Ca2þ homeostasis and the molecular pathways expressed at a higher level than is calreticulin, it regulates. Overexpressing calreticulin within its Ca2þ binding should be considered low ca- the lumen of the ER increases the amount of pacity, approximately 1–2 mol of Ca2þ per mol Ca2þ that can be released after inhibition of of protein, and low affinity (Lievremont et al. SERCAvia thapsigargin, showing that calreticu- 1997; Lamb et al.