Golgi pH and Ion Homeostasis in Health and Disease.

Maintenance of the main Golgi functions, glycosylation and sorting, is dependent on the unique Golgi pH microenvironment that is thought to be set by the balance between the rates of V-ATPase-mediated proton pumping and its leakage back to the cytoplasm via an unknown pathway. The concentration of other ions, such as chloride, potassium, calcium, magnesium, and manganese, is also important for Golgi homeostasis and dependent on the transport activity of other ion transporters present in the Golgi membranes. During the last decade, several new disorders have been identified that are caused by, or are associated with, dysregulated Golgi pH and ion homeostasis. Here, we will provide an updated overview on these disorders and the proteins involved. We will also discuss other disorders for which the molecular defects remain currently uncertain but which potentially involve proteins that regulate Golgi pH or ion homeostasis.

A dominant negative splice variant of the heparan sulfate biosynthesis enzyme NDST1 reduces heparan sulfate sulfation.

NDST1 (glucosaminyl N-deacetylase/N-sulfotransferase) is a key enzyme in heparan sulfate (HS) biosynthesis, where it is responsible for HS N-deacetylation and N-sulfation. In addition to the full length human enzyme of 882 amino acids, here designated NDST1A, a shorter form containing 825 amino acids (NDST1B) is synthesized after alternative splicing of the NDST1 mRNA. NDST1B is mostly expressed at a low level, but increased amounts are seen in several types of cancer where it is associated with shorter survival. In this study, we aimed at characterizing the enzymatic properties of NDST1B and its effect on HS biosynthesis. Purified recombinant NDST1B lacked both N-deacetylase and N-sulfotransferase activities. Interestingly, HEK293 cells overexpressing NDST1B synthesized HS with reduced sulfation and altered domain structure. Fluorescence resonance energy transfer-microscopy demonstrated that both NDST1A and NDST1B had the capacity to interact with the HS copolymerase subunits EXT1 and EXT2 and also to form NDST1A/NDST1B dimers. Since lysates from cells overexpressing NDST1B contained less NDST enzyme activity than control cells, we suggest that NDST1B works in a dominant negative manner, tentatively by replacing the active endogenous NDST1 in the enzyme complexes taking part in biosynthesis.

SLC4A2 anion exchanger promotes tumour cell malignancy via enhancing net acid efflux across golgi membranes.

Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive "H+ leak" pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, reverse their invasive and anchorage-independent growth phenotype. These findings suggest a possibility to return malignant cells to a benign state by restoring Golgi resting pH.

Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains.

ß-1,4-Galactosyltransferase 1 (B4GALT1) and ST6 ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) catalyze the successive addition of terminal ß-1,4-linked galactose and α-2,6-linked sialic acid to N-glycans. Their exclusive interaction in the Golgi compartment is a prerequisite for their full catalytic activity, whereas a lack of this interaction is associated with cancers and hypoxia. To date, no structural information exists that shows how glycosyltransferases functionally assemble with each other. Using molecular docking simulations to predict interaction surfaces, along with mutagenesis screens and high-throughput FRET analyses in live cells to validate these predictions, we show here that B4GALT1 and ST6GAL1 interact via highly charged noncatalytic surfaces, leaving the active sites exposed and accessible for donor and acceptor substrate binding. Moreover, we found that the assembly of ST6GAL1 homomers in the endoplasmic reticulum before ST6GAL1 activation in the Golgi utilizes the same noncatalytic surface, whereas B4GALT1 uses its active-site surface for assembly, which silences its catalytic activity. Last, we show that the homomeric and heteromeric B4GALT1/ST6GAL1 complexes can assemble laterally in the Golgi membranes without forming cross-cisternal contacts between enzyme molecules residing in the opposite membranes of each Golgi cisterna. Our results provide detailed mechanistic insights into the regulation of glycosyltransferase interactions, the transitions between B4GALT1 and ST6GAL1 homo- and heteromers in the Golgi, and cooperative B4GALT1/ST6GAL1 function in N-glycan synthesis.

N-acetylglucosaminyltransferases and nucleotide sugar transporters form multi-enzyme-multi-transporter assemblies in golgi membranes in vivo.

Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.

Dermatan sulfate epimerase 1 and dermatan 4-O-sulfotransferase 1 form complexes that generate long epimerized 4-O-sulfated blocks.

During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid-containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid-forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.

Crystal structures of eukaryote glycosyltransferases reveal biologically relevant enzyme homooligomers.

Glycosyltransferases (GTases) transfer sugar moieties to proteins, lipids or existing glycan or polysaccharide molecules. GTases form an important group of enzymes in the Golgi, where the synthesis and modification of glycoproteins and glycolipids take place. Golgi GTases are almost invariably type II integral membrane proteins, with the C-terminal globular catalytic domain residing in the Golgi lumen. The enzymes themselves are divided into 103 families based on their sequence homology. There is an abundance of published crystal structures of GTase catalytic domains deposited in the Protein Data Bank (PDB). All of these represent either of the two main characteristic structural folds, GT-A or GT-B, or present a variation thereof. Since GTases can function as homomeric or heteromeric complexes in vivo, we have summarized the structural features of the dimerization interfaces in crystal structures of GTases, as well as considered the biochemical data available for these enzymes. For this review, we have considered all 898 GTase crystal structures in the Protein Data Bank and highlight the dimer formation characteristics of various GTases based on 24 selected structures.

LTCC Packaged Ring Oscillator Based Sensor for Evaluation of Cell Proliferation.

A complementary metal-oxide-semiconductor (CMOS) chip biosensor was developed for cell viability monitoring based on an array of capacitance sensors utilizing a ring oscillator. The chip was packaged in a low temperature co-fired ceramic (LTCC) module with a flip chip bonding technique. A microcontroller operates the chip, while the whole measurement system was controlled by PC. The developed biosensor was applied for measurement of the proliferation stage of adherent cells where the sensor response depends on the ratio between healthy, viable and multiplying cells, which adhere onto the chip surface, and necrotic or apoptotic cells, which detach from the chip surface. This change in cellular adhesion caused a change in the effective permittivity in the vicinity of the sensor element, which was sensed as a change in oscillation frequency of the ring oscillator. The sensor was tested with human lung epithelial cells (BEAS-2B) during cell addition, proliferation and migration, and finally detachment induced by trypsin protease treatment. The difference in sensor response with and without cells was measured as a frequency shift in the scale of 1.1 MHz from the base frequency of 57.2 MHz. Moreover, the number of cells in the sensor vicinity was directly proportional to the frequency shift.

Glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized.

Glycosylation is the most common and complex cellular modification of proteins and lipids. It is critical for multicellular life and its abrogation often leads to a devastating disease. Yet, the underlying mechanistic details of glycosylation in both health and disease remain unclear. Partly, this is due to the complexity and dynamicity of glycan modifications, and the fact that not all the players are taken into account. Since late 1960s, a vast number of studies have demonstrated that glycosyltransferases typically form homomeric and heteromeric complexes with each other in yeast, plant and animal cells. To propagate their acceptance, we will summarize here accumulated data for their prevalence and potential functional importance for glycosylation focusing mainly on their mutual interactions, the protein domains mediating these interactions, and enzymatic activity changes that occur upon complex formation. Finally, we will highlight the few existing 3D structures of these enzyme complexes to pinpoint their individual nature and to emphasize that their lack is the main obstacle for more detailed understanding of how these enzyme complexes interact and function in a eukaryotic cell.

UDP-sugar substrates of HAS3 regulate its O-GlcNAcylation, intracellular traffic, extracellular shedding and correlate with melanoma progression.

Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.

Fluorescence resonance energy transfer (FRET) and proximity ligation assays reveal functionally relevant homo- and heteromeric complexes among hyaluronan synthases HAS1, HAS2, and HAS3.

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

Organizational interplay of Golgi N-glycosyltransferases involves organelle microenvironment-dependent transitions between enzyme homo- and heteromers.

Glycosylation of proteins and lipids takes place in the Golgi apparatus by the consecutive actions of functionally distinct glycosidases and glycosyltransferases. Current evidence indicates that they function as enzyme homomers and/or heteromers in the living cell. Here we investigate their organizational interplay and show that glycosyltransferase homomers are assembled in the endoplasmic reticulum. Upon transport to the Golgi, the majority of homomers are disassembled to allow the formation of enzyme heteromers between sequentially acting medial-Golgi enzymes GnT-I and GnT-II or trans-Golgi enzymes GalT-I and ST6Gal-I. This transition is driven by the acidic Golgi environment, as it was markedly inhibited by raising Golgi luminal pH with chloroquine. Our FRAP (fluorescence recovery after photobleaching) measurements showed that the complexes remain mobile Golgi membrane constituents that can relocate to the endoplasmic reticulum or to the scattered Golgi mini-stacks upon brefeldin A or nocodazole treatment, respectively. During this relocation, heteromers undergo a reverse transition back to enzyme homomers. These data unveil an unprecedented organizational interplay between Golgi N-glycosyltransferases that involves dynamic and organelle microenvironment-driven transitions between enzyme homomers and heteromers during their trafficking within the early secretory compartments.

Enrichment of sweat-derived extracellular vesicles of human and bacterial origin for biomarker identification.

Sweat contains biomarkers for real-time non-invasive health monitoring, but only a few relevant analytes are currently used in clinical practice. In the present study, we investigated whether sweat-derived extracellular vesicles (EVs) can be used as a source of potential protein biomarkers of human and bacterial origin. Methods: By using ExoView platform, electron microscopy, nanoparticle tracking analysis and Western blotting we characterized EVs in the sweat of eight volunteers performing rigorous exercise. We compared the presence of EV markers as well as general protein composition of total sweat, EV-enriched sweat and sweat samples collected in alginate skin patches. Results: We identified 1209 unique human proteins in EV-enriched sweat, of which approximately 20% were present in every individual sample investigated. Sweat derived EVs shared 846 human proteins (70%) with total sweat, while 368 proteins (30%) were captured by medical grade alginate skin patch and such EVs contained the typical exosome marker CD63. The majority of identified proteins are known to be carried by EVs found in other biofluids, mostly urine. Besides human proteins, EV-enriched sweat samples contained 1594 proteins of bacterial origin. Bacterial protein profiles in EV-enriched sweat were characterized by high interindividual variability, that reflected differences in total sweat composition. Alginate-based sweat patch accumulated only 5% proteins of bacterial origin. Conclusion: We showed that sweat-derived EVs provide a rich source of potential biomarkers of human and bacterial origin. Use of commercially available alginate skin patches selectively enrich for human derived material with very little microbial material collected.

Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells.

Glycosylation is one of the most common modifications of proteins and lipids and also a major source of biological diversity in eukaryotes. It is critical for many basic cellular functions and recognition events that range from protein folding to cell signaling, immunological defense, and the development of multicellular organisms. Glycosylation takes place mainly in the endoplasmic reticulum and Golgi apparatus and involves dozens of functionally distinct glycosidases and glycosyltransferases. How the functions of these enzymes, which act sequentially and often competitively, are coordinated to faithfully synthesize a vast array of different glycan structures is currently unclear. Here, we investigate the supramolecular organization of the Golgi N- and O-glycosylation pathways in live cells using a FRET flow cytometric quantification approach. We show that the enzymes form enzymatically active homo- and/or heteromeric complexes within each pathway. However, no complexes composed of enzymes that operate in different pathways, were detected, which suggests that the pathways are physically distinct. In addition, we show that complex formation is mediated almost exclusively by the catalytic domains of the interacting enzymes. Our data also suggest that the heteromeric complexes are functionally more important than enzyme homomers. Heteromeric complex formation was found to be dependent on Golgi acidity, markedly impaired in acidification-defective cancer cells, and required for the efficient synthesis of cell surface glycans. Collectively, the results emphasize that the Golgi glycosylation pathways are functionally organized into complexes that are important for glycan synthesis.

Altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc defines the highly invasive cancer cell phenotype.

Several distinct metastasis-associated glycosylation changes have been shown to promote cancer cell invasion and metastasis, the main cause of death of cancer patients. However, it is unclear whether their presence reflects cell- or tissue-specific variations for metastasis, or species needed to drive different phases of the metastatic cascade. To address this issue from a different perspective, we investigated here whether different cancer cell lines share any glycotopes that are common and important for their invasive phenotype. By using lectin microarray glycan profiling and an established myoma tissue-based 3D invasion assay, we identified a single glycotope recognized by Helix Pomatia agglutinin (HPA), whose expression level in different cancer cells correlated significantly with their invasive potential. Lectin pull-down assay and LC-MS/MS analysis in highly- (A431 and SW-48) and poorly invasive (HepG2 and RCC4) cancer cells revealed ~85 glycoproteins of which several metastasis-promoting members of the integrin family of cell adhesion receptors, the epidermal growth factor receptor (EGFR) and the matrix metalloproteinase-14 (MMP-14) were among the abundant ones. Moreover, we showed that the level of the GalNAc glycotope in MMP-14, EGFR, αV-, ß1- and ß4 integrin in highly and poorly invasive cancer cells correlated positively with their invasive potential. Collectively, our findings suggest that altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc drives the highly invasive cancer cell phenotype.

Golgi N-glycosyltransferases form both homo- and heterodimeric enzyme complexes in live cells.

Glycans (i.e. oligosaccharide chains attached to cellular proteins and lipids) are crucial for nearly all aspects of life, including the development of multicellular organisms. They come in multiple forms, and much of this diversity between molecules, cells, and tissues is generated by Golgi-resident glycosidases and glycosyltransferases. However, their exact mode of functioning in glycan processing is currently unclear. Here we investigate the supramolecular organization of the N-glycosylation pathway in live cells by utilizing the bimolecular fluorescence complementation approach. We show that all four N-glycosylation enzymes tested (beta-1,2-N-acetylglucosaminyltransferase I, beta-1,2-N-acetylglucosaminyltransferase II, 1,4-galactosyltransferase I, and alpha-2,6-sialyltransferase I) form Golgi-localized homodimers. Intriguingly, the same enzymes also formed two distinct and functionally relevant heterodimers between the medial Golgi enzymes beta-1,2-N-acetylglucosaminyltransferase I and beta-1,2-N-acetylglucosaminyltransferase II and the trans-Golgi enzymes 1,4-galactosyltransferase I and alpha-2,6-sialyltransferase I. Given their strict Golgi localization and sequential order of function, the two heterodimeric complexes are probably responsible for the processing and maturation of N-glycans in live cells.

N-Glycan Biosynthesis: Basic Principles and Factors Affecting Its Outcome.

Carbohydrate chains are the most abundant and diverse of nature's biopolymers and represent one of the four fundamental macromolecular building blocks of life together with proteins, nucleic acids, and lipids. Indicative of their essential roles in cells and in multicellular organisms, genes encoding proteins associated with glycosylation account for approximately 2% of the human genome. It has been estimated that 50-80% of all human proteins carry carbohydrate chains-glycans-as part of their structure. Despite cells utilize only nine different monosaccharides for making their glycans, their order and conformational variation in glycan chains together with chain branching differences and frequent post-synthetic modifications can give rise to an enormous repertoire of different glycan structures of which few thousand is estimated to carry important structural or functional information for a cell. Thus, glycans are immensely versatile encoders of multicellular life. Yet, glycans do not represent a random collection of unpredictable structures but rather, a collection of predetermined but still dynamic entities that are present at defined quantities in each glycosylation site of a given protein in a cell, tissue, or organism.In this chapter, we will give an overview of what is currently known about N-glycan synthesis in higher eukaryotes, focusing not only on the processes themselves but also on factors that will affect or can affect the final outcome-the dynamicity and heterogeneity of the N-glycome. We hope that this review will help understand the molecular details underneath this diversity, and in addition, be helpful for those who plan to produce optimally glycosylated antibody-based therapeutics.

Ae2(a,b)-deficient mice exhibit osteopetrosis of long bones but not of calvaria.

Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pH(i)) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na(+)-independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2(a,b)(-/-) mice (deficient in the main isoforms Ae2a, Ae2b(1), and Ae2b(2)). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2(a,b)(-/-) mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2(a,b)(-/-) mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts--but not long-bone osteoclasts--possess a sodium-dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2(a,b)(-/-) mice.

Loss of USF2 promotes proliferation, migration and mitophagy in a redox-dependent manner.

The upstream stimulatory factor 2 (USF2) is a transcription factor implicated in several cellular processes and among them, tumor development seems to stand out. However, the data with respect to the role of USF2 in tumor development are conflicting suggesting that it acts either as tumor promoter or suppressor. Here we show that absence of USF2 promotes proliferation and migration. Thereby, we reveal a previously unknown function of USF2 in mitochondrial homeostasis. Mechanistically, we demonstrate that deficiency of USF2 promotes survival by inducing mitophagy in a ROS-sensitive manner by activating both ERK1/2 and AKT. Altogether, this study supports USF2's function as tumor suppressor and highlights its novel role for mitochondrial function and energy homeostasis thereby linking USF2 to conditions such as insulin resistance, type-2 diabetes mellitus, and the metabolic syndrome.

Elevated Golgi pH impairs terminal N-glycosylation by inducing mislocalization of Golgi glycosyltransferases.

Acidic pH of the Golgi lumen is known to be crucial for correct glycosylation, transport and sorting of proteins and lipids during their transit through the organelle. To better understand why Golgi acidity is important for these processes, we have examined here the most pH sensitive events in N-glycosylation by sequentially raising Golgi luminal pH with chloroquine (CQ), a weak base. We show that only a 0.2 pH unit increase (20 microM CQ) is sufficient to markedly impair terminal alpha(2,3)-sialylation of an N-glycosylated reporter protein (CEA), and to induce selective mislocalization of the corresponding alpha(2,3)-sialyltransferase (ST3) into the endosomal compartments. Much higher pH increase was required to impair alpha(2,6)-sialylation, or the proximal glycosylation steps such as beta(1,4)-galactosylation or acquisition of Endo H resistance, and the steady-state localization of the key enzymes responsible for these modifications (ST6, GalT I, MANII). The overall Golgi morphology also remained unaltered, except when Golgi pH was raised close to neutral. By using transmembrane domain chimeras between the ST6 and ST3, we also show that the luminal domain of the ST6 is mainly responsible for its less pH sensitive localization in the Golgi. Collectively, these results emphasize that moderate Golgi pH alterations such as those detected in cancer cells can impair N-glycosylation by inducing selective mislocalization of only certain Golgi glycosyltransferases.