High Ca2+ content in the Golgi apparatus (Go) is essential for protein processing and sorting. In addition, the Go can shape the cytosolic Ca2+ signals by releasing or sequestering Ca2+. We generated two new aequorin-based Ca2+ probes to specifically measure Ca2+ in the cis/cis-to-medial-Go (cGo) or the trans-Go (tGo). Ca2+ homoeostasis in these compartments and in the endoplasmic reticulum (ER) has been studied and compared. Moreover, the relative size of each subcompartment was estimated from aequorin consumption. We found that the cGo accumulates Ca2+ to high concentrations (150–300 μM) through the sarco plasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). The tGo, in turn, is divided into two subcompartments: tGo1 and tGo2. The subcompartment tGo1 contains 20% of the aequorin and has a high internal [Ca2+]; Ca2+ is accumulated in this subcompartment via the secretory pathway Ca2+-ATPase 1 (SPCA-1) at a very high affinity (K50=30 nM). The subcompartment tGo2 contains 80% of aequorin, has a lower [Ca2+] and no SPCA-1 activity; Ca2+ uptake happens through SERCA and is slower than in tGo1. The two tGo subcompartments, tGo1 and tGo2, are diffusionally isolated. Inositol trisphosphate mobilizes Ca2+ from the cGo and tGo2, but not from tGo1, whereas caffeine releases Ca2+ from all the Golgi regions, and nicotinic acid dinucleotide phosphate and cADP ribose from none.
- calcium transport
- endoplasmic reticulum
- Golgi apparatus
- trans-Golgi apparatus
Abbreviations: cADPR, cADP ribose; cGo, cis-Golgi apparatus; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced GFP; ER, endoplasmic reticulum; GA, EGFP–aequorin; GM130, 130-kDa Golgi matrix protein; Go, Golgi apparatus; HSV, herpes simplex virus; InsP3, inositol 1,4,5-trisphosphate; LAMP2, lysosome-associated membrane protein 2; NAADP, nicotinic acid-adenine dinucleotide phosphate; RyR, ryanodine receptor; SERCA, sarco plasmic/endoplasmic reticulum Ca2+-ATPase; SPCA, secretory pathway Ca2+-ATPase; TBH, 2,5-di(t-butyl)-1,4-benzohydroquinone; TGN46, 46-kDa trans-Golgi network protein; tGo, trans-Go
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