Ca²⁺-dependent liquid-liquid phase separation underlies intracellular Ca²⁺ stores

Abstract

Endoplasmic/sarcoplasmic reticulum Ca2+ stores are essential to myriad cellular processes, however, the structure of these stores is largely unknown and existing models neither explain observations made in vivo nor sufficiently account for physiological data. We investigate CASQ1 - the major Ca2+ binding protein of skeletal muscle - and discover Ca2+-dependent liquid-liquid phase separation activity. The intrinsic disorder of CASQ1 underlies this activity and is regulated via phosphorylation by the secretory pathway kinase FAM20C. This novel divalent cation driven condensation demonstrates liquid-liquid phase separation occurs within the endoplasmic/sarcoplasmic reticulum, mechanistically explains efficient Ca2+ buffering and storage, and represents a largely unexplored mechanism of divalent-cation driven protein association.

The Role of Intrinsically Disordered Proteins in Liquid–Liquid Phase Separation during Calcium Carbonate Biomineralization

“…Less is known concerning the ability of polyanionic proteins to undergo LLPS in a similar charge-dependent manner. Mayfield et al identified a previously unknown mechanism of calcium-dependent LLPS occurring within the endoplasmic/sarcoplasmic reticulum (ER/SR) that explains efficient calcium ion buffering and storage [ 44 ]. It was shown that calcium ions modulate LLPS of the polyanionic protein and major calcium-binding protein of the SR of skeletal muscle, calsequestrin-1 (CASQ1).…”

“…CASQ1 is an IDP that influences its capacity for LLPS. It was also shown that the LLPS of CASQ1 is regulated via phosphorylation by the secretory pathway kinase Fam20C, which phosphorylates structurally-conserved regions of CASQ1 [ 44 ]. Thus, the phosphorylated protein (pCASQ1) more readily entered the LLPS state.…”

“…Thus, the phosphorylated protein (pCASQ1) more readily entered the LLPS state. Mayfield et al [ 44 ] suggested that this likely arises from the increased disorder and conformational flexibility of pCASQ1. Additionally, they hypothesized that calcium-dependent LLPS of polyanionic IDPs is a widespread and evolutionarily-conserved phenomenon that might represent a major mechanism underlying calcium ion handling and signaling [ 44 ].…”

The Structural–Functional Crosstalk of the Calsequestrin System: Insights and Pathological Implications

“…Imaging in vivo and cross-linking experiments support the notion that this material is Ca 2+ -complexed CASQ [18][19][20]. In addition, these structures disassemble and disperse under luminal Ca 2+ depletion conditions [15,21], which correlates with the fact that the recombinant protein can organize in vitro into multiple architectures of varying compactness, hierarchically ordered in response to rising Ca 2+ concentrations from micromolar levels up to 20 mM [3,16,17,22] (Figure 2). As higher order polymeric structures are formed, the capacity and cooperativity of Ca 2+ -binding events show a parallel, stepwise increase, which has consequences for the modulation of intra-SR Ca 2+ storage and release.…”

Insight into Calcium-Binding Motifs of Intrinsically Disordered Proteins

“…IDPs often engage in multivalent interactions [60], including those leading to liquid-liquid phase separation [61][62][63]. Emerging research shows that phase separation can be regulated by calcium in the endoplasmic/sarcoplasmic reticula to enable efficient calcium buffering [64]. Taken together, this suggests that the negatively charged IDPs may work to sponge calcium towards the multi-protein complexes, thus regulating calcium bioavailability and signaling.…”