The early Golgi compartments host many Ca²⁺ pumps that are also highly abundant in the ER, such as Sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCAs), possibly because of the constant content exchange between the Golgi and ER, mediated by direct contact and vesicles ^[2][3][28,29]. On the other hand, the Golgi also contains unique Ca²⁺ transporters not found in the ER, including Secretory pathway Ca²⁺-transporting ATPases (SPCAs, also known as the Calcium-transporting ATPase type 2C members) and TMEM165 ^[3][29]. Different from ER Ca²⁺ pumps, they usually show high affinity to Mn²⁺ ion and therefore act as Mn²⁺ transporters as well.

It is widely accepted that SERCAs and SPCAs both contribute to the Ca²⁺ uptake into the Golgi since it is only partially inhibited by the SERCA inhibitor thapsigargin ^[4][5][38,39]. Isolated membranes show enrichment of SERCA in the ER and cis-Golgi while SPCA1 in the trans-Golgi membranes ^[6][40]. This conclusion was supported by immunoelectron microscopy (immuno-EM) ^[7][41]. Additionally, investigation using Ca²⁺ probes targeted to the cis- and trans-Golgi showed that the Ca²⁺ accumulation in these compartments is mainly facilitated by SERCA and SPCA1, respectively ^[5][39]. As Golgi-specific Ca²⁺ pumps, SPCAs have been shown to be the major Ca²⁺ pumps that facilitate the Ca²⁺ uptake in the trans-Golgi network (TGN).

TMEM165 was first identified in congenital disorders of glycosylation (CDG) ^[8][17] and localize to the Golgi ^[9][42]. Loss of TMEM165 function interrupts Ca²⁺, pH, and Mn²⁺ homeostasis and causes glycosylation abnormalities, which are restored by Mn²⁺ supplementation. Therefore, TMEM165 is proposed to be a Golgi-localized Ca²⁺/Mn²⁺ antiporter involved in glycosylation regulation ^[10][11][43,44]. Interestingly, TMEM165 is relocated to lysosomes and subjected to lysosomal degradation upon high Mn²⁺ exposure in a Rab7 and Rab5 independent manner, indicating a Mn²⁺-dependent TMEM165 quality control mechanism involving direct targeting of the protein to the lysosomal degradation pathway ^[12][45]. The Mn²⁺-induced translocation and lysosomal degradation of TMEM165 was later associated to the function of SPCA1, since the phenotype was rescued by a gain-of-function mutation of SPCA1. This suggests a possible functional link between the two proteins in response to the cytosolic Mn²⁺ change, however the underlying mechanism remains unclear ^[13][46]. One of the unanswered questions is why TMEM165 is degraded upon high Mn²⁺ exposure. One proposal is that TMEM165 works as a Mn²⁺ importer using the gradient of Golgi Ca²⁺. TMEM165 degradation would increase cytosolic Mn²⁺ and engage the major Mn²⁺ importer SPCA1 for detoxification. Another hypothesis is that TMEM165 imports Ca²⁺ into the Golgi using the Golgi Mn²⁺ gradient. Then TMEM165 degradation would restrain the Mn²⁺ in the Golgi to avoid further toxication. Therefore, TMEM165 is proposed to function in both directions depending on the combined effects of Ca²⁺ and Mn^{2+ [12]}[45].

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs) are two major types of Ca²⁺-release channels in the Golgi. They both open at low cytosolic Ca²⁺ and close at high cytosolic Ca²⁺ concentrations. IP3R receptors are encoded by 3 genes (ITPR1, 2 and 3) in human, and are found in Golgi membranes ^[2][14][28,47]. These proteins are better characterized as ER-resident Ca²⁺ channels that release Ca²⁺ from the ER lumen into the cytosol in response to the stimulation of IP3 ^[15][9]. The RyR family members encoded by 3 genes (RYR1, 2 and 3) in humans are the other major types of Ca²⁺ channels that mediate the release of Ca²⁺ into the cytoplasm ^[16][48]. The IP3Rs were once considered to be the only Golgi Ca²⁺ channels, since IP3 but not RyR agonists significantly induced Ca²⁺ release from Golgi membrane fractions in the presence of thapsigargin ^[17][49]. However, other experiments in rat sympathetic neurons and neonatal cardiac myocytes argued that functional RyRs are found in the trans-Golgi and probably take part in Golgi Ca²⁺ release ^[18][19][50,51]. Emetine, an alkaloid, induces Ca²⁺ release from trans-Golgi without inducing Ca²⁺ release from the ER. It is unlikely that emetine induces Ca²⁺ release via activation of RyR2 or inhibition of SPCA, and the underlying molecular mechanism requires further investigation ^[20][52].

In addition to the Ca²⁺ transporters and release channels summarized above, Ca²⁺-binding proteins are another factor involved in Ca²⁺ homeostasis in the Golgi lumen. CALNUC, 45 kDa calcium-binding protein (Cab45), p54/NEFA, and calumenin are identified as major Ca²⁺ binding proteins found specifically in the Golgi lumen. CALNUC (also known as Nucleobindin-1), an EF-hand, Ca²⁺-binding peripheral membrane protein closely linked with the lumenal surface of the cis-Golgi cisternae, is the most abundant and well-characterized Golgi Ca²⁺ binding protein ^[21][53]. Transiently expressed CALNUC co-distributes with IP3R1 in cells, which increases Ca²⁺ storage as well as Ca²⁺ release upon ATP or IP3 stimulation ^[14][47]. Overexpression of SPCA upregulates the expression of CALNUC, indicating that CALNUC, together with SERCA and IP3R, is involved in establishment of the agonist-mobilizable Golgi Ca²⁺ store.

Cab45 is the first ubiquitous Golgi Ca²⁺-binding protein identified. It is a soluble Golgi lumen-resident protein that binds Ca²⁺ via its EF-hands ^[22][54]. Retention of Cab45 in the TGN is Ca²⁺-dependent, which is disrupted by treatment of cells with a Ca²⁺ ionophore ^[23][55], and is regulated by the Golgi resident serine/threonine kinase Fam20C ^[24][56]. Once bound to Ca²⁺, Cab45 oligomerizes and is recruited by secretory proteins. Via super resolution microscopy, the Cab45 oligomer-cargo complex is detected to be preferentially colocalized with the TGN Ca²⁺ pump SPCA1. Phosphorylation of Cab45 by Fam20C reduces the size of Cab45 oligomers, abolishes the TGN retention, and enhances the sorting and secretion of Cab45-cargo complex ^[24][56]. These results suggest the possibility that a functional sorting complex formed by Cab45, SPCA1, and cargo protein is regulated by Golgi Ca^{2+ [25]}[57].

p54/NEFA ^[26][58] and calumenin are two other Ca²⁺-binding proteins found in the Golgi lumen. p54/NEFA is a medial Golgi membrane-associated lumenal protein sharing sequence homology with CALNUC ^[27][59]. Calumenin is a glycosylated secretory protein distributed throughout the secretory pathway with a low affinity to Ca²⁺ ions ^[28][29][60,61]. It is reported to interact with RyR2 and SERCA in cardiac and skeletal sarcoplasmic reticulum, and therefore suggested to be a regulator of RyR2 and SERCA, which is tightly coupled with Ca²⁺ cycling ^[30][31][32][62,63,64].

In summary, the Golgi is equipped with all the molecular components necessary for maintaining Ca²⁺ homeostasis in the Golgi lumen: Ca²⁺ ATPases that pump Ca²⁺ into the Golgi lumen, Ca²⁺ channels that release Ca²⁺ into the cytoplasm, and Ca²⁺-binding proteins that buffer Ca²⁺ in the Golgi lumen. With all these regulators in action, the distinct sub-compartments of the Golgi appear to have a decreasing lumenal Ca²⁺ concentration through heterogenous expression of Ca²⁺ transporters and channels: the cis-Golgi expresses mainly SERCA and IP3Rs and contains around 250 µM lumenal Ca²⁺; the medial Golgi mainly expresses SERCA and SPCA1; and the trans-Golgi mainly expresses SPCA1 and RyRs, with a lumenal Ca²⁺ of about 130 µM (Figure 1) ^[3][29].

Inhibition of Ca²⁺ pumps was reported to trigger a Golgi morphology change. Knockdown of SPCA1 leads to Golgi ribbon fragmentation, shortened and tubulated cisternae, and complete absence of cis- and trans-Golgi compartments ^[7][41]. In a separate study, transient inhibition of SERCA by thapsigargin-induced Golgi fragmentation before the ER unfolded protein response (UPR) is triggered, indicating an ER-stress-independent Golgi morphology disruption ^[33][65]. Further experiments demonstrated that thapsigargin-induced elevation of cytosolic Ca²⁺ activates protein kinase C (PKCα), which subsequently phosphorylates and inactivates GRASP55, a key membrane tether in Golgi stack formation ^[34][35][66,67]. Golgi structural defects also impact membrane trafficking and secretion. Activation of PKCα with phorbol 12-myristate 13-acetate (PMA) and histamine modulates Golgi structure in a similar fashion, indicating a link between Ca²⁺ signaling, Golgi structure and function, and human physiology ^[36][37].

Many Golgi-resident glycosylation enzymes such as Golgi α-mannosidases, and proteases such as furin, show Ca²⁺-binding activity ^[37][68] or undergoes Ca²⁺-dependent cleavage, which is required for their activation ^[38][69]. Taking the Golgi mannosidases as examples, these cis-Golgi resident enzymes are required in the early steps of N-glycosylation process, removing mannose residues from a high-mannose intermediate product. Ca²⁺ binds to mannosidases prior to the substrate and remains associated during the enzymatic reaction. Divalent cations, including Mn²⁺, compete with Ca²⁺ and therefore inhibit the mannosidase activity ^[39][70].

Mn²⁺ is also a common ion associated with many Golgi-residing glycosylation enzymes, including N-acetylglucosaminyltransferases ^[40][71], N-acetylgalactosaminyltransferases ^[41][72], mannosyltransferases ^[42][73], and some members in the fucosyltransferase family ^[43][44][74,75]. While Mn²⁺ homeostasis in the Golgi lumen is required for proper glycosylation, high cytosolic Mn²⁺ is toxic and related to many human diseases (Table 1) ^[45][46][76,77]. By uptaking Mn²⁺ from cytoplasm into the Golgi lumen, the SPCA pumps avoid cytotoxic accumulation of Mn²⁺, therefore maintain Golgi Mn²⁺ homeostasis and accurate glycosylation ^[47][48][49][78,79,80]. On the other hand, exposure of Mn²⁺ protects cells from some bacteria-originated toxins (e.g., Shiga toxin, STx) through down-regulation of Golgi phosphoprotein of 130 kDa (GPP130) and protects against STx-induced death ^[50][81]. As a type 2 ribosome inactivating protein, STx binds to the cellular toxin receptor to be internalized, and is then transported via the retrograde trafficking route through endosomes and Golgi to the ER, where the toxin is translocated to the cytosol to practice its toxin activity ^[51][52][82,83]. GPP130 serves as a host-cell trafficking receptor that facilitates the intake of the toxin into the TGN via the interaction with Syntaxin 5 ^[53][84]. Exposure to Mn²⁺ blocks the retrograde trafficking of STx from endosome to Golgi and leads to its lysosomal degradation ^[50][81]. Mn²⁺ targets GPP130 and induces its oligomerization, causing GPP130 redistributed to lysosomes via a clathrin and Rab7-dependent mechanism, where it is subsequently degraded ^[54][55][56][85,86,87]. Because of its sensitivity to Mn²⁺ exposure, GPP130 can be used as an intra-Golgi Mn²⁺ sensor in Golgi Mn²⁺ homeostasis studies ^[49][80].

The role of Ca²⁺ in trafficking was first indicated in a study on ER-to-Golgi trafficking in 1989 ^[79][88]. Since then, reports on Ca²⁺ participating in intracellular transport events have emerged ^{[80][81][82][83]}[89,90,91,92] and Ca²⁺ have been shown to be a fundamental factor in intracellular trafficking ^[84][93]. Incubation of isolated Golgi membranes with ionomycin and thapsigargin, which reduce the Golgi lumenal Ca²⁺, inhibits intra-Golgi transport of VSV-G ^[85][94]. On the other hand, high Ca²⁺ concentration in the TGN is required for the segregation and sorting of secretory cargo at TGN. This involves the Ca²⁺ pump SPCA1 and Ca²⁺ binding protein Cab45 ^{[23][86][87][88]}[55,95,96,97]. SPCA1 binds to the actin-severing protein actin-depolymerizing factor (ADF)/cofilin1 on the TGN via dynamic actin and promotes Ca²⁺ influx into the TGN lumen. Increased Ca²⁺ leads to Cab45 oligomerization and binding to the secretory cargo proteins. The Cab45-cargo complex is then sorted into sphingomyelin-rich vesicles and transported to the plasma membrane for secretion, which is fine-tuned by the Golgi lumenal kinase Fam20C ^[89][98]. Knockdown of either SPCA1 or ADF/cofilin1 results in significant mis-sorting of secretory cargo proteins, indicating the role of actin-filaments-induced SPCA1 activation and Ca²⁺ uptake at TGN in protein sorting ^[87][96].

Given the Golgi’s role in protein post-translational modification and sorting, the importance of lumenal Ca²⁺/Mn²⁺ in controlling many of the enzymatic activities within the Golgi, as well as the effect of Ca²⁺/Mn²⁺ fluctuation on the Golgi structure, any changes in Ca²⁺/Mn²⁺ homeostasis are likely to have an impact on Golgi function. Changes in Golgi Ca²⁺/Mn²⁺ handling should, in turn, result in significant changes in cellular activities, contributing to disease pathogenesis ^[90][99]. Indeed, mutations in Ca²⁺/Mn²⁺ transporters and channels have been related to various diseases affecting multiple organs and tissues characterized with a wide range of clinical features (summarized in Table 1). In addition, SPCA1 is reported to be required for the maturation and spread of diverse viruses. SPCA1 deficiency leads to impaired virus glycoprotein maturation, reduces the infectivity of furin-requiring viruses, and lowers viral burden in human airway epithelial cells possibly by decreasing the abundance of furin ^[91][100]. Importantly, the Spike protein of SARS-CoV-2 virus, which is responsible for the strong infectivity of the virus, contains a furin cleavage site, indicating furin activity as a potential therapeutic target of the wide-spreading COVID-19 ^[92][101].