Golgi pH, Ion and Redox Homeostasis: How Much Do They Really Matter?

1Compartmentalization is a key feature of eukaryotic cells, and it allows cells to complete various tasks with amazing speed and specificity. One drawback to compartmentalization is its need for the extra energy required to generate the unique luminal environments of each compartment. Unlike the ER, the other secretory pathway compartments, such as the ERGIC, Golgi apparatus and secretory vesicles, are all mildly acidic. Each of them has its own unique resting pH (pH set point) that facilitates their efficient functioning, be it membrane trafficking, cargo selection, glycosylation, proteolysis, or protein sorting. Nevertheless, these compartments also have common properties with the ER, with which they all communicate at least to some extent. The ER and the Golgi apparatus are the most closely associated compartments, sharing several common properties including high calcium concentration and oxidative potential, as well as the ability to synthesize glycans. This review, focusing on the Golgi apparatus, will discuss ER-related processes for comparative purposes only.

2In general, organelle acidity is driven by the ATP-mediated proton pump, the V(vacuolar)-ATPase, which is counterbalanced by anion influx or cation efflux, and proton leak back to the cytoplasm via a “H+ leak channel” whose identity still remains elusive. Many other energy consuming pumps and leak channels, which are needed to maintain balanced Cl-, Ca2+, Mn2+ and K+ levels, are also present in the Golgi membranes. These include the Golgi pH regulator (GPHR, a chloride channel), a mid-1-related chloride channel (MClC) and voltage-gated chloride channels ClC-3B in mammalian cells, and Gef-1 in yeast. Golgi membranes also contain two different isoforms of the Na+/H+ exchanger (NHE7 and NHE8), of which NHE7 seems to mediate the influx of Na+ or K+ in exchange for H+. Although the exact physiological roles of many of these transporters remain unclear, they are known to contribute to Golgi resting pH, membrane potential, vesicular trafficking and protein sorting in the organelle. The use of fluorescent redox probes has recently revealed the Golgi redox state to be important for Golgi homeostasis and functions. showed that the oxidative potential of the Golgi is higher than that of the endoplasmic reticulum (ER), the main site of disulfide bond formation in the cells. Indeed, earlier observations had shown that disulfide bonds can also form “late,” i.e., in the Golgi compartment, and facilitate disulfide bond-mediated oligomerization of some secretory products before their secretion to the extracellular space. Cellular oxygen levels also regulate the level of Golgi nitric oxide (NO), a free radical that has been shown to be important for maintaining Golgi morphology and for ensuring continued membrane trafficking to the cell surface. Taken together, these examples highlight the complexity of factors needed to maintain the unique Golgi environment and the functions that depend on it. The following paragraphs summarize the molecular machineries involved, before focusing on why their failures result in organelle dysfunction and disease.

3The acidity of the Golgi lumen was first demonstrated in 1983 by using electron microscopy and a compound (DAMP) that accumulated in acidic cellular compartments. Currently, several different fluorescence-based approaches have been used to identify proteins that contribute to Golgi acidity and its resting pH. These studies have shown that different Golgi sub-compartments have distinct pH set points, decreasing along the cis–trans axis of the Golgi stack from pH 6.7 (cis-Golgi) to pH 6.0 at the trans-Golgi network (TGN). A pertinent question is how this pH gradient is established and maintained along the Golgi stack, given its dynamic nature resulting from the continuous flow of incoming and leaving vesicular carriers. Another related issue is whether a similar gradient also applies to other ions that are uniquely concentrated in the Golgi lumen and thus contribute to its ion homeostasis.

4The resting pH of the Golgi lumen is now known to be determined mainly by three different ion transport systems that include the vacuolar (V)-ATPase-mediated proton pump, counter ion (Cl-) transport, and proton “leak” across the Golgi membranes back to the cytoplasm. In brief, the V-ATPase uses ATP as an energy source to pump protons into the Golgi lumen. Due to proton pumping, the membrane potential starts to increase (inside positive) and must be counterbalanced by Cl- influx. This very likely takes place via the GPHR Cl- channel (or a cation efflux channel). Once the Golgi pH is sufficiently acidic (pH < 6.3), proton efflux via an elusive “proton leak channel” prevents further acidification of the Golgi lumen. The resting pH, or the pH set point, is established once the rate of proton pumping matches its leak rate across Golgi membranes. Because of continuous H+ pumping by the V-ATPase, it is the rate of H+ leakage that dictates the resting pH of the organelle. The authors showed that the rate of proton efflux decreases between successive secretory compartments. They also suggested that the higher density of H+ pumps in the later secretory compartments may also contribute to their lower resting pH. These two factors are likely responsible also for the decreasing pH gradient along the cis- to trans-axis of the Golgi stacks, even though direct proof for this does not yet exist.

5The V-ATPase itself is a multi-subunit protein complex, whose composition may vary between different compartments. For example, the Golgi localized V-ATPase appears to possess a subunit a different from that found in other V-ATPases (the Stv1p instead of the Vph1p in yeast). The V-ATPase activity is also regulated by glucose or nutrient levels, yet under normal conditions (i.e., at least when counter-ion conductance is sufficient and, therefore, does not restrict proton pumping), it is assumed to be constantly active. In support of this, the Golgi lumen in intact cells starts to alkalinize when the V-ATPase activity is shut down by using concanamycin A (Figure 1, green dots).

6Cl- influx seems to be normally required to prevent membrane potential increase due to proton pumping by the V-ATPase. Under normal conditions, it is considered to be high enough and mediated by the GPHR protein channel termed the Golgi pH Regulator. Mutation of the protein was shown to increase Golgi resting pH (by 0.4–0.5 pH units), alter glycosylation, delay transport to the plasma membrane, and induce Golgi fragmentation. These findings thus provide strong support for the view that H+ pumping is dependent on Cl- influx and is needed to maintain a constant membrane potential. The extent to which other Golgi-localized chloride channels, such as the voltage-gated chloride channels ClC-3B and Gef1p in yeast regulate Golgi resting pH remains unclear.

7Other studies have suggested that continuous H+ pumping may be facilitated by passive K+ efflux rather than by Cl- influx. This may relate to a high permeability of the Golgi membranes to K+ ions, and could perhaps be mediated by Na+ and K+ conductive channels or transporters such as the Na+/K+-ATPase. In support of the latter possibility, acetylstrophanthidin (an inhibitor of the Na+/K+-ATPase) was proposed to increase luminal acidity by inhibiting electrogenic Na+/K+ exchange (3 Na+ for 2 K+), thereby reducing the accumulation of other cations (relative to H+) in the Golgi lumen. Alternatively, the Na+/H+ exchanger NH7 could also facilitate the acidification of the Golgi lumen by transporting H+ into the Golgi lumen in exchange for luminal K+ ions. However, recent data indicates that NH7 does not transport K+ ions, thus leaving open whether Na+ ions may suffice for an acid loading function of this exchanger in the Golgi compartment.

8Despite its importance, the identity of the “proton leak channel” still remains elusive. It may involve exchange of luminal protons for cytosolic cations via a proton conductive channel, or via import of base equivalents. Physiological measurements indicate that proton efflux in the TGN is voltage-sensitive and inhibited by Zn2+, suggesting the involvement of a regulated channel. Other studies suggest that the molecular characteristics of a putative H+ channel mimic those of the plasma membrane H+ channels. Therefore, the two ubiquitously expressed, Golgi-localized Na+/H+ exchanger isoforms, NHE7 and NHE8, are good candidates for this channel, because Na+/H+ exchange is normally driven by existing ion gradients, and high amounts of H+ in the Golgi lumen will drive influx of Na+. In support of this, overexpression of both NHE7 and NHE8 were found to increase Na+ and K+ influx to the Golgi lumen, and both also raised the Golgi resting pH. However, changes in sodium concentration during Golgi pH measurements did not markedly alter the Golgi resting pH, leaving some doubts about the possible roles of these NHEs in mediating proton leakage across Golgi membranes. In accordance with this, recently showed that NHE7, at least when expressed at the plasma membrane, acts as an acid loader rather than as a “H+ leak” pathway in the cells. The NHE8 isoform also seems to have more pronounced effects on endosomes than it has on the Golgi. Therefore, further work is needed to clarify the exact roles of the NHEs in the Golgi membranes.

9Soluble buffering molecules may also be used for regulating the Golgi resting pH. In support of this view, a homolog of the erythrocyte anion exchanger 1 (Band 3, AE1, SLC4A1) was identified as the AE2a isoform (SLC4A2a) of the SLC4A gene family in the Golgi membranes in a number of cell types. All members of this gene family are electroneutral HCO3-/Cl- exchangers, regulating cytosolic pH, chloride concentration and cell volume through an obligatory one to one exchange of chloride for bicarbonate. Our recent high-throughput Golgi pH measurements indicate that it is involved in Golgi pH regulation, as its overexpression increased, and knockdown decreased, the Golgi resting pH (Khosrowabadi et al., unpublished). The potential involvement of various channels, pumps and transporters in the maintenance of Golgi homeostasis is summarized in Figure 2.

10Ca2+, Mg2+, or Mn2+ ions are present at high concentrations in the Golgi lumen. Their presence is important for cargo concentration and sorting and glycosylation. Golgi membranes also possess relevant pumps for Ca2+ and Mn2+ uptake (SERCA2, SPCA1/2), channels for Ca2+ release (IP3R, RyR) and luminal proteins that bind Ca2+ with high affinity. Mn2+ ions are important cofactors for Golgi-resident glycosyltransferases. The DXD motif conserved in many glycosyltransferases appears to have a key role in Mn2+-mediated donor substrate binding and catalytic activity. Mn2+ ions also act as scavengers for reactive oxygen species (ROS). Of these, SERCA and SPCA type pumps are responsible for the maintenance of the low cytosolic and high luminal Ca2+ concentrations typical of many secretory pathway compartments. These two Ca2+ pumps seem to contribute differentially to Ca2+ uptake into the Golgi, as SERCA2 is enriched in the cis-Golgi, while SPCA1 is mainly present in the trans-Golgi. The localization of Ca2+ release channels, the inositol-1,3,5-trisphosphate receptor (IP3Rs) and the ryanodine receptor (RyR), also seems to be different, as IP3 did not release Ca2+ in the trans-Golgi, while activation by caffeine did so. Contrasting with SERCAs, SPCAs are also engaged in Mn2+ transport, and thus can provide this essential trace metal supply to Golgi glycosyltransferases.

11Recent evidence also suggests that mutations of TMEM165 cause a type II congenital disorder of glycosylation in humans by interfering with Mn2+ and, perhaps, also Ca2+/H+ transport and, therefore, also with Golgi ion and pH homeostasis and glycosylation. However, it is not yet fully clear what role this multi-spanning membrane protein plays in Golgi ion homeostasis. Recent evidence indicates that, unlike the Golgi-localized SPCA1, the ER-associated SERCA pump 2b isoform partially rescued TMEM165 KO-induced glycosylation defect by Mn2+. Moreover, those authors also recently showed that the TMEM165 KO can also be rescued by galactose supplement in HEK293 cell culture media, or when given to patients. Further studies are needed to reveal the exact role of the TMEM165 transporter in the maintenance of Golgi ion homeostasis.

12Another multi-spanning membrane protein capable of transporting Ca2+ has been identified in the Golgi membranes. This protein, named Golgi-associated anti-apoptotic protein (GAAP), has recently gained increasing attention due to its role in tumorigenesis. It is a member of the Transmembrane Bax Inhibitor-1 Motif-containing (TMBIM) protein family regulating Ca2+ levels and fluxes in intracellular stores, confering resistance to a broad range of apoptotic stimuli and promoting cell adhesion and migration via the activation of store-operated Ca2+ entry (SOCE).

13The Golgi lumen also harbors several Ca2+-binding proteins, including Cab45, CALNUC, p54/NEFA and calumenin, all of which, except for Cab45, are distinct from their ER counterparts. Of these, the most abundant is CALNUC, an EF-hand, Ca2+-binding protein resident in the CGN and cis-Golgi cisternae. It plays a major role in Ca2+ buffering and secretion through the Golgi. Recent studies of Cab45 have demonstrated its importance in Ca2+-mediated protein sorting. Cab45 is the core component of this oligomerization-driven sorting mechanism, also involving the cytoplasmic actin cytoskeleton, and the Ca2+ ATPase SPCA1. The system relies first on the local synthesis of sphingomyelin at the TGN membrane enhancing Ca2+ import by SPCA1, which then drives secretory protein sorting and export, thereby coupling lipid synthesis to protein sorting and secretion.

14Genetically encoded and targeted fluorescent probes such as roGFP and HyPer have been used to determine organelle redox states. By using roGFP2 as a probe, we have previously shown that mitochondria have a less oxidizing environment than that of the ER. Intriguingly, it was also found that the Golgi lumen is more oxidizing than the ER despite being considered as the most oxidizing compartment in eukaryotic cells. One possibility for its higher oxidizing power is that it serves for “late” disulfide bond formation, as indicated in studies showing that the assembly of von Willebrand factor oligomers to multimers, or other secretory products, requires tail-to-tail disulfide bond formation in the Golgi. Such disulfide bond formation in the Golgi is likely assisted by members of the Quiescin-Sulfhydryl Oxidase (QSOX) gene family that all display PDI-like thioredoxin (Trx) domains and ERV-like oxidase domains. These domains allow QSOX proteins to efficiently couple disulfide bond formation with the reduction of molecular oxygen to hydrogen peroxide. QSOX1 is widely expressed within the secretory pathway compartments, while one of the splice variants, QSOX1a, mainly localizes to the Golgi. This suggests its involvement in disulfide bond formation, possibly related to the maturation of ECM components, or to the formation of higher order structures in the Golgi. Other proteins that can regulate Golgi redox homeostasis include the glutaredoxins Grx6 and Grx7. They both belong to an ubiquitous family of proteins that catalyze the reduction of disulfide bonds with the help of reduced glutathione. Grx6 and Grx7 represent the first glutaredoxins found in the cis-Golgi in baker’s yeast. They both show a high glutaredoxin activity in vitro, and yeast cells lacking both proteins exhibit growth defects and a strongly increased sensitivity toward oxidizing agents. Grx6 and Grx7 are probably important for counteracting oxidation-driven disulfide bond formation in the Golgi.

15The availability of oxygen is also intimately linked to the Golgi redox state. When low, it causes hypoxia, a condition that affects multiple cellular compartments including mitochondria and the ER. Recent evidence indicates that hypoxia also modulates Golgi functions and, in particular, those related to membrane trafficking and glycosylation events. Accordingly, hypoxia has been shown to alter expression levels of both glycosyltransferase and nucleotide sugar transporter genes, and to inhibit membrane trafficking between the ER and the Golgi. We recently showed that hypoxia modulates the Golgi redox state and glycosylation without markedly affecting Golgi pH homeostasis (; see also below). Oxygen levels not only affect the redox state of the Golgi lumen, but also the production of NO levels by modulating the activity and expression of various NO synthase isoforms, including neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). Of these, only eNOS is located to the Golgi membranes via myristylation or palmitylation of its N-terminus. NO is a lipophilic compound and can readily pass through membranes. Both NO and superoxide (O2-), another possible product of eNOS activity, are highly reactive free radicals and increase ROS load.

16Failure to maintain Golgi pH, ion, and redox homeostasis is commonly associated with membrane trafficking and protein sorting defects. Monensin, a Na+/H+ ionophore, was the first compound shown to block intra-Golgi transport between the medial- and trans-Golgi cisternae. reported that intracellular transport of the Uukuniemi virus membrane glycoproteins (G1 and G2) was not inhibited by monensin. Whether this discrepant behavior is cell or virus type-dependent remains unclear. Protein sorting in the Golgi is also dependent upon existing pH gradients. In line with these observations, showed that monensin induces the relocalization of B4GalT1 galactosyltransferase (but not ST6Gal-I) and alpha-1,3-fucosyltransferase 6 in swollen vesicles derived from the TGN based on their colocalization with TGN46, a specific TGN marker. This relocalization was also found to be signal-mediated, involving a short sequence in its cytoplasmic tail, which, when present in ST6Gal-I, was able to relocate the latter into the TGN-derived swollen vesicles from the trans-Golgi cisternae or the TGN. However, the signals were not needed for the steady state localization of these enzymes in the trans-Golgi cisternae.

17A better example of pH-sensitive membrane trafficking steps is the retrograde transport from the Golgi back to the ER. This was demonstrated using bafilomycin A (a V-ATPase specific inhibitor), which inhibited retrograde, but not anterograde, transport from the intermediate compartment (IC)/cis-Golgi back to the ER. This preferential effect on retrograde trafficking may relate to the more acidic environment at the IC/cis-Golgi interface than that of the ER and to the pH-dependent retrieval system mediated by the KDEL-receptor (see below). Based on these observations, it seems that cargo selection and membrane fission is more sensitive to a pH change than membrane fusion is, thereby inhibiting or delaying transport between successive secretory compartments., However, due to the pH-sensitivity of viral protein-mediated membrane fusion events with endosomal membranes, this scenario needs further testing. Indeed, these viral fusion events typically occur via “inside to outside” fusions with organelle membranes and are therefore topologically opposite from fusions that take place between vesicular carriers and their target membranes.

18Another well-characterized example of a pH-dependent protein sorting step is the KDEL receptor, which returns escaped ER resident proteins from the cis-Golgi back to the ER. The receptor is a key component of a homeostatic control system that regulates trafficking between the ER and the Golgi compartments and within the Golgi itself. The receptor binds the peptide sequence KDEL (or a similar sequence motif), leading to interaction with two different Golgi-associated heterotrimeric G-proteins, which regulate the transport machineries via phosphorylation. Of notice here is that both cargo binding and its release are regulated by the pH gradient between the two organelles (see, and references therein). In the more acidic environment of the cis-Golgi, the receptor recognizes the motif and binds to it, while at the neutral pH of the ER lumen, it releases the motif and the associated cargo. Moreover, p58/ERGIC-53/LMAN1, a receptor protein involved in the export of soluble glycoproteins from the ER, employs a similar pH gradient for its oligomerization and accessory protein-mediated binding with specific cargo and its release in the low pH-high calcium environment at the ER-Golgi interface.

19A third well-known example is the mannose-6-phosphate receptor, which binds lysosomal enzymes carrying the man-6-P tag in the Golgi and releases them in the lower pH environment of the endosomes. In line with this, we showed that, in some cancer cell lines with problems in lysosomal acidification, the ligand-bound receptor cannot unload its ligand in lysosomes and accumulates in endosomal/lysosomal compartments. This suggested that further lysosomal enzyme cargo sorting at the TGN is impossible and can result in their aberrant secretion into the extracellular space, a phenomenon that is often associated with tumorigenesis and likely helps cancer cells to invade and metastasize to adjacent tissues.

20Golgi pH homeostasis is also important for the sorting of apical and basolateral proteins in polarized epithelial cells. showed that laminin and heparan sulfate proteoglycan (HSPG) are normally actively sorted to the basolateral surface of polarized canine renal tubule cells (MDCK) in a pH-dependent manner. By increasing the pH of the Golgi and other cellular compartments in MDCK cells with NH4Cl (Figure 1, red dots), the authors were able to divert the two above-mentioned secretory proteins to both the apical and basolateral transport vesicles, with the outcome that roughly equal amounts were sorted to both surfaces in the treated cells. Since the TGN is the main sorting station for these two surface domains, it is likely that the cargo recognition and sorting at the TGN may not depend only on specific sorting signals but also on the existence of an environment suitable for their recognition by the sorting machinery in each case. We recently showed that the apical targeting of the CEAMCAM5 (carcinoembryonic antigen, CEA), a well-known follow-up marker for colorectal cancer, is also a pH-sensitive process. CEA is a typical GPI-anchored apical protein present in gut epithelial cells. For an unknown reason, CEA exhibits a non-polarized distribution in cancer cells, such as in CaCo-2 cells. Guided by the notion that the Golgi resting pH is ∼0.5 pH units higher in CaCo-2 cells than in non-malignant cells, we treated MDCK cells stably expressing CEA with various compounds, including concanamycin A (CMA: a proton pump inhibitor, see Figure 1). We showed that, in contrast to drugs affecting the redox state, CMA attenuated apical targeting of CEA without affecting its trafficking to the cell surface. In the presence of the drug, CEA was delivered equally to apical and basolateral domains of MDCK cells due to inhibition of its GPI anchor-mediated association with lipids rafts.

21Autosomal recessive Cutis Laxa type II is the first inherited disease identified thus far that is tightly linked to altered Golgi pH homeostasis. The skin of these patients shows excessive wrinkling at an early age. It is caused by mutations in the gene encoding the a2 subunit of the Golgi-localized V-ATPase (ATP6V0A2). However, patients belonging to a closely related disease group suffering from Wrinkly skin syndrome are heterogeneous, in that only some patients carry the same mutation and show no symptoms of elastin deficiency. Although the Golgi resting pH has not yet been directly measured, it is expected that this V-ATPase defect perturbs Golgi pH homeostasis, because the patients’ cells exhibited glycosylation and membrane trafficking defects. Further studies are still needed, as Golgi membranes also seem to co-express another subunit, the “a1” isoform, of the V-ATPase (see ). Nevertheless, the “a2” subunit mutations impair retrograde trafficking from the Golgi back to the ER, but here again, the mechanistic details remain unclear. One complicating factor in gaining an understanding of the cutis laxa phenotype is the fact that the “a2” subunit appears to localize also in early endosomes, suggesting that altered endosomal pH and dysfunction may also contribute to the disease etiology.

22recently described a new pH homeostasis-associated disease with multigenerational non-syndromic intellectual disability (ID). The disease is caused by missense mutations in the alkali cation/proton exchanger NHE7 (SLC9A7). The variant protein localized correctly in the TGN/post-Golgi vesicles, but its N-linked glycans were abnormal likely due to less acidic pH of the TGN/post-Golgi compartments in patient’s cells. Membrane trafficking, however, was unaffected. These observations are consistent with a role for NHE7 in the regulation of TGN/post-Golgi pH homeostasis and suggest that abnormal Golgi pH homeostasis may be the cause of neurodevelopmental defects associated with this disease.

23Other luminal ions, and particularly Ca2+, also contribute to membrane trafficking defects. The depletion of cellular Ca2+ stores in NRK cells using thapsigargin abolished KDEL receptor-mediated retrieval of ER chaperones GRP94/endoplasmin and GRP78/BiP, resulting in their appearance in the culture medium. Accordingly, thapsigargin was found to inhibit Brefeldin A-induced retrograde transport from the Golgi back to the ER in HeLa cells. Calcium depletion also selectively inhibited proteolytic cleavage of pro-somatostatin or proinsulin, without affecting their secretion. It also interfered with the sorting of secretogranin II into immature granules in semi-intact PC12 cells, even though high Ca2+ and low pH have been reported to facilitate the concentration of cargo proteins in regulated secretory vesicles.

24SPCA1 is a Golgi-localized Ca2+ ATPase that transports both Ca2+ and Mn2+ into the Golgi lumen and, therefore, plays an important role in Golgi cation homeostasis. In humans, allelic mutations of the SPCA1 gene are the cause of Hailey–Hailey disease, in which patients’ keratinocytes exhibit an increased cytosolic Ca2+ concentration, and defects in protein sorting and Ca2+ signaling. Lowered levels of Ca2+ and Mn2+ cations in the Golgi lumen in patients’ cells lead to defects in protein folding, trafficking and sorting or proteolytic cleavage of prohormones. These defects could explain why the affected patients are unable to maintain structurally intact desmosomes and epidermis. The fact that Mn2+ is an important cofactor for many glycosyltransferases suggests that glycosylation is altered in affected cells and may also contribute to the disease etiology.

25Golgi Ca2+ (and Mn2+) homeostasis is also dependent on cellular oxygen levels. A good example of this is the fact that intermittent hypoxia upregulates the expression of both SPCAs in HCT116 cells, suggesting that Ca2+ and/or Mn2+ transport from the cytosol to the Golgi lumen via SPCAs likely increases in hypoxic cells. Oxygen also regulates nitrogen oxide (NO) levels in the Golgi by modulating eNOS activity and thus, NO production, thereby locally enhancing S-nitrosylation of Golgi proteins, especially of the N-ethylmaleimide-sensitive factor (NSF). Since NSF is involved in membrane fusion events, this modification delays protein transport from the ER to the plasma membrane and, thus, can at least partially explain why hypoxia inhibits ER-Golgi vesicular trafficking. On the other hand, compounds that can scavenge NO (such as c-PTIO, N-acetylcysteine and hemoglobin) induced Golgi fragmentation, which was accompanied by the depletion of α-soluble NSF acceptor protein (α-SNAP) from Golgi membranes, in accordance with the observed delay in ER-Golgi trafficking.

26Glycosylation is likely the most pH-sensitive process of the Golgi functions. For example, monensin was shown to prevent processing of Uukuniemi viral G proteins into endo-H-resistant and under-sialylated species without affecting membrane trafficking. in turn were able induce the expression of oncofetal Thomsen-Friedenreich (TF- or T-) antigen in LS174T goblet-differentiated cells by increasing Golgi pH with bafilomycin A and monensin. were the first to provide a mechanistic link for these pH-induced glycosylation changes by using prolonged NH4Cl treatment in HeLa and LS 174T cells. They showed that inhibition of O-glycan synthesis by NH4Cl was accompanied by mis-localization of N-acetylgalactosaminyltransferase 2, b-1,2-N-acetylglucosaminyltransferase I and b-1,4-galactosyltransferase 1 into endosomal compartments, while the drug had no effect on Golgi morphology. However, because most of the enzymes that elongate O-glycans were not addressed in the study, it remains unclear whether enzyme re-localization is solely responsible for the observed glycosylation defect(s). Later, by using increasing concentrations of chloroquine, we demonstrated that only a 0.2 pH unit increase in Golgi luminal pH is needed to interfere with mucin type O-glycosylation and terminal a-2,3-sialylation of N-linked glycans without causing any changes to overall Golgi morphology. The latter defect correlated well with the observed mislocalization of the relevant sialyltransferase (ST3Gal-III) into endosomal compartments, while no such redistribution was observed with ST6Gal-I (or B4GalT-I), i.e., the enzyme that adds sialic acid to terminal galactose residue via an a-2,6-linkage. Cutis laxa type II patients also display defects in sialylation of both N-linked and O-linked glycans. These observations indicate that glycosylation in general is highly sensitive to changes in Golgi luminal pH and, if altered, can be due to mislocalization of a selected set of glycosyltransferases. Other known causes are changes in the expression levels of the enzymes, yet these do not strictly correlate with glycan profiles displayed by the cells.

27Most glycosyltransferases show an intrinsic tendency to form oligomeric complexes with each other. Typically, such complexes include enzymes that successively add sugar residues to a glycan chain. All enzymes also form homomers in the ER, perhaps facilitating their folding or transport to the Golgi, or both. On the other hand, enzyme heteromers only form after the enzymes arrive in the Golgi compartment. This switch from enzyme homomers to enzyme heteromers is dependent on the pH gradient or Golgi redox homeostasis (see section “Golgi Redox Homeostasis and Altered Glycosylation” last paragraph) between the ER and the Golgi. Thus, heteromer formation of N-glycan processing enzymes B4GalT-I and ST3Gal-III, and of enzymes that synthesize mucin type O-glycan core structures (ppGalNacT-6, C1GalT-1, C2/3GNT), is prevented by increasing Golgi pH with chloroquine. Intriguingly, the same enzymes (except B4GalT-I) were also found to mislocalize in chloroquine treated cells (unpublished observations), suggesting that heteromer formation may contribute to their retention or retrieval in the Golgi membranes. However, in other cases such as B4GalT-I and ST6Gal-I, heteromer formation was not affected by an increase in Golgi luminal pH, but rather by an altered Golgi redox state (; see also Section “Golgi Redox Homeostasis and Altered Glycosylation” last paragraph). These observations point to fundamental differences in the way enzyme heteromers form in the Golgi lumen, and perhaps reflecting the high specificity of the interactions needed to prevent irrational interactions that could otherwise lead to the synthesis of mixed or irrelevant glycan structures. The high specificity for the interactions could also explain a failure to identify any consensus Golgi retention motif(s) in Golgi enzymes, except those needed for their retrieval from later compartments to earlier ones via GOLPH3-mediated binding to the COPI complex. Whether such Golgi retention motifs involving relevant enzyme interactions in different Golgi sub-compartments indeed exist remains to be tested. However, their existence is supported by the pH-dependent mislocalization of a set of glycosyltransferases (; unpublished observations); otherwise, it would be difficult to understand how luminal alkalization can interfere with the recognition of retrieval motifs on the cytosolic side of the Golgi membranes. In addition, oligomerization that inherently involves enzyme interactions, has been considered to be important for the retention of resident glycosyltransferases in the Golgi.

28The loss of enzyme heteromers is generally accompanied by changes in O- and N-linked glycosylation, but likely concerns other glycosylation pathways as well. One reason for these changes is that heteromer formation significantly increases the activity of the complex constituents. For example, both enzyme activities of B4GalT-I/ST6Gal-I heteromers were 2.5-fold higher than their respective homodimers. How this activation is achieved is currently unclear but may involve substrate channeling or conformational changes brought about by the interaction. In other words, the formation of the enzyme heteromers from enzyme homomers in the Golgi could simply serve to keep enzymes silent until they arrive in the Golgi. Such a system would increase both the speed and fidelity of glycan synthesis, as it would also prevent the intervention of competing enzymes that can use the same acceptor sugar as a substrate. This view is in line with the pH-independent α-2,6-sialylation and formation of B4GalT-I/ST6Gal-I heteromers (in contrast to α-2,3-sialylation and the formation of ST3Gal-III/B4GalT-I heteromers; see; ). The difference in pH-dependency can also explain why the carcinoembryonic antigen (CEACAM5) extracted from colon cancer tissue carries α-2,6-linked sialic acid instead of the α-2,3-linked sialic acid found in normal tissues. The extent to which the loss of enzyme heteromers contributes to glycosylation remains to be determined when enzyme interaction mutants become available.

29Altered glycosylation is one of the hallmarks of cancers. Such alterations can involve changes in the elongation of O-glycans, the branching of N-glycans, sulfation, O-acetylation of sialic acid, fucosylation and the expression of blood group antigens. Some of these changes are used as cancer markers, while others also have verified roles in promoting tumorigenesis. Based on existing data, several factors that can cooperatively contribute to the above cancer-associated glycosylation changes have been put forward. These include the altered expression of glycosyltransferases or nucleotide sugar transporter genes, and perhaps also a loss of activity of Cosmc, a specific molecular chaperone needed for the folding and catalytic activation of C1GalT-I. The C1GalT-I enzyme normally adds galactose to the Tn-antigen (GalNAc-Ser), forming a mucin type O-glycan core 1 structure (the T-antigen). Thus, the loss of its activity may result in an increased expression of the Tn-antigen in cancer cells.

30Cancer-associated glycosylation changes can also result from a more general defect related to altered Golgi ion or pH homeostasis. The first observations suggesting this came from studies in which the treatment of cells with pH gradient dissipating drugs increased the expression of cancer-associated Tn- and T-antigens. At high concentrations, these same compounds also induced Golgi fragmentation typically seen also in cancer cells. Direct Golgi pH measurements with fluorescent probes in breast and colorectal cancer cells (MCF-7, HT-29, SW-48) showed that the Golgi resting pH is indeed more alkaline (∼0.2–0.4 pH units) than that of non-malignant cells. These early observations strongly supported the view that abnormally high Golgi resting pH is responsible for the increased expression of cancer-associated glycan antigens.

31An important question at the time was why abnormal Golgi pH is detrimental to glycosylation. Although one still cannot exclude possible effects on the synthesis or transport of nucleotide sugars, we believe that either the loss of the O-glycosyltransferase heteromers or enzyme mislocalization, or both, are the two main reasons for the pH-dependent glycosylation changes seen in cancer cells. These two factors might in fact also be interlinked, given that oligomerization appears to be important for Golgi retention. Thus, at elevated Golgi resting pH, the enzymes responsible for synthetizing the O-glycan core structure are unable to form heteromers. Their loss then abrogates their retention in the Golgi, whereby they mislocalize to endosomal compartments and are therefore unable to elongate the core GalNAc residue with other sugar residues in the Golgi. In support of this view, O-glycosyltransferases seem to have altered distribution in cancer cells in vivo. Similar relocalization of the initiating ppGalNAcT-1/2 to the ER was seen after growth factor-induced activation of the src kinase, or by transfecting cells with constitutively active src. This relocalization was linked to a COP-I-dependent trafficking event, as a dominant-negative Arf1 isoform, Arf1(Q71L), blocked ppGalNacT redistribution. Note however, that the pH-induced relocalization of the enzyme involved transport to the endosomal compartments via bulk flow, suggesting that pH-induced relocalization of the enzymes is associated with impaired Golgi retention, rather than with activated transport from the Golgi to the ER.

32Another issue related to organelle acidification defects in cells is its association with the multidrug resistance (MDR). In certain MDR cancer cell lines, chemotherapeutic drugs (often weak bases) become protonated and sequestered in acidic organelles. Sequestration in resistant cells allows the removal of cytotoxic drugs from the cytoplasm via secretory and recycling pathways. In contrast, drug-sensitive cells were shown to have defects in organelle acidification, whereby a similar sequestration of the drugs does not occur, exposing the cells to high concentrations of the drugs. However, these acidification defects are not universal in all MDR cells, suggesting that other mechanisms for MDR exist.

33Reactive oxygen species and hypoxia (low oxygen environment) are key modulators of the cellular redox state. ROS and hypoxia also modulate Golgi-associated vesicular trafficking, protein sorting and glycosylation events. Most often, this is thought to be mediated mainly by hypoxia-inducible factors (HIF-1-3) that regulate the expression of hundreds of genes, including a variety of proteins involved in glycosylation. Specifically, hypoxia has been shown to down- or up-regulate enzymes that synthetize nucleotide sugars in the cytoplasm, Golgi-localized glycosyltransferases (Mgat2, Mgat-3 and Mgat5 and 5b, fucosyltransferases 1, 2 and 7, sialyltransferases ST3Gal-I and ST6Gal-1) and transporters of UDP-Galactose, CMP-sialic acid (Sialin) and UDP-N-acetylglycosamine (UGT1). Some of these are involved in the synthesis of cancer-associated sialyl Lewis A/X carbohydrate epitopes typically found in selectins on O-linked glycans and glycolipids. HIF-1α in the Pa-Tu-8988S and Pa-Tu-8988T pancreatic cancer cell lines have also been shown to suppress the expression of the UDP-glucuronosyltransferase, cytosolic O-GlcNAc transferase (OGT) and glucosylceramide synthase (GCS). Finally, showed that expression of the SPCA2 Ca2+ pump in HCT116 colon cancer cells was upregulated by hypoxia, and by reactive oxygen and nitrogen species. The authors suggested that this upregulation is associated with Mn2+-dependent cell cycle arrest, but whether these changes relate to increased Ca2+ or Mn2+ transport to the Golgi lumen, Ca2+-mediated protein sorting, glycosylation, or detoxification from excess Mn2+, remains unclear.

34Based on the above observations, Taniguchi et al. introduced the term “Glyco-redox” to link altered glycosylation with oxidative stress generated by hypoxia or ROS, and to emphasize their close association with Parkinson disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and chronic obstructive pulmonary disease (COPD). These changes may also partly involve cleavage of cell surface glycosaminoglycans and N-linked glycans, thereby affecting interactions of cells with the extracellular matrix, as has been observed using hypoxia-mimicking agents such as CoCl2. Oxidative stress also provides a link between altered glycosylation, high-fat diet and the onset of type II diabetes. High levels of free fatty acids were shown to inhibit the activity of two transcription factors (Foxa2 and Hnf1a) that normally positively regulate the expression of Mgat4a, a glycosyltransferase needed for β1,4-GlcNAc branching of N-glycans. This modification is needed for insulin-stimulated transport of the GLUT-4 transporter to the cell surface, whereby it is able to bind and import glucose for further use by the pancreatic β-cells. In the absence of β1,4-GlcNAc branching, the GLUT-4 transporter remains intracellular, leading to an accumulation of glucose in the blood.

35We have recently shown that hypoxia also modulates glycosylation in a HIF-independent manner by reducing Golgi redox state. Specifically, we demonstrated that even moderate hypoxia (5% O2) lowers the Golgi oxidizing potential to the level found in the ER of normoxic cells, consistent with possible problems in disulfide bond formation. Based on lectin microarray glycan profiling, this decrease was accompanied by an attenuated sialylation of N-glycans, an elongation of O-linked glycans, the loss of pH-independent interaction between the B4GalT-I and ST6Gal-I, and the loss of ST6Gal-I activity in hypoxic cells. These findings can explain the reduced a-2,6-sialylation in hypoxic cells. Given that sialylation was not the only change in glycosylation, we expect that other enzymes are similarly affected by a lowered Golgi oxidizing potential.

36The above examples highlight the critical roles of Golgi pH, ion and redox homeostasis in the maintenance of Golgi functions and its unique architecture. The existing data also emphasizes that Golgi acidity, high cation concentrations and redox state are not just background components in the Golgi, but rather, they emerge as the key players in orchestrating various membrane trafficking events, keeping the Golgi resident enzymes correctly localized and active, and facilitating their cooperative interactions in glycosylation. In addition, they are important for cargo selection by post-Golgi vesicular carriers and for protein sorting to the apical surface in polarized epithelial cells. Further support of their importance is provided by several human diseases, including Cutis Laxa, Hailey–Hailey disease and congenital disorder of glycosylation 2K (CDG2K), which all are caused by altered ion homeostasis in the Golgi lumen. Other Golgi homeostasis-associated diseases are certainly waiting to be identified.

37Although many mechanistic details remain incompletely understood, the current evidence indicates that each of the Golgi pH, ion or redox (oxygen) regulatory systems can perturb all Golgi functions simultaneously or may specifically impair only one of them. For example, a small increase in Golgi luminal pH (0.2 pH units) will perturb glycosylation with no detectable effect on other Golgi functions. A failure to maintain Golgi oxidative potential causes defects in both membrane trafficking and glycosylation, with no detectable changes in enzyme localization in the Golgi. High Ca2+ concentrations seem to contribute to membrane trafficking and protein sorting events at the Golgi, but also to glycosylation, since many enzymes need Mn2+ ions to remain catalytically active.

38The above examples highlight the complexity and mutual interplay of the regulatory systems needed to establish and maintain Golgi homeostasis and the many different cellular phenotypes encountered by manipulating homeostatic machineries with drugs or mutations. For example, it is well known that perturbed membrane trafficking often results in changes in Golgi architecture and glycosylation. The inter-dependence of these phenomena is probably the biggest obstacle for better understanding the role of each of these regulatory systems in the Golgi. Nevertheless, such studies are needed to provide us with new insights into how the various Golgi tasks are executed and to identify the molecular machineries that act in the background to keep these tasks ongoing. These studies will eventually unveil how the Golgi compartment functions as an organelle and what purpose(s) its unique architecture with stacked and flattened cisternae actually serves for.

39The datasets generated for this study are available on request to the corresponding author.

40SK wrote the text and made the figures.

41The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.