The gradual acidification of the secretory pathway is conserved and extremely important for eukaryotic cells, but until now there was no pH sensor available to monitor the pH of the early Golgi apparatus in Saccharomyces cerevisiae. Therefore, we developed a pHluorin-based sensor for in vivo measurements in the lumen of the Golgi. By using this new tool we show that the cis- and medial-Golgi pH is equal to 6.6-6.7 in wild type cells during exponential phase. As expected, V-ATPase inactivation results in a near neutral Golgi pH. We also uncover that surprisingly Vph1p isoform of the V-ATPase is prevalent to Stv1p for Golgi acidification. Additionally, we observe that during changes of the cytosolic pH, the Golgi pH is kept relatively stable, mainly thanks to the V-ATPase. Eventually, this new probe will allow to better understand the mechanisms involved in the acidification and the pH control within the secretory pathway.

Institute of Physiology Czech Academy of Sciences CZ 14220 Prague Czech Republic

Louvain Institute of Biomolecular Science and Technology Université catholique de Louvain B 1348 Louvain la Neuve Belgium

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Schonichen A, Webb BA, Jacobson MP, Barber DL. Considering protonation as a posttranslational modification regulating protein structure and function. Annual review of biophysics. 2013;42:289–314. doi: 10.1146/annurev-biophys-050511-102349. PubMed DOI PMC

Sorensen SO, van den Hazel HB, Kielland-Brandt MC, Winther JR. pH-dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro. European journal of biochemistry/FEBS. 1994;220:19–27. doi: 10.1111/j.1432-1033.1994.tb18594.x. PubMed DOI

Van Den Hazel H, Wolff AM, Kielland-Brandt MC, Winther JR. Mechanism and ion-dependence of in vitro autoactivation of yeast proteinase A: possible implications for compartmentalized activation in vivo. The Biochemical journal. 1997;326(Pt 2):339–344. doi: 10.1042/bj3260339. PubMed DOI PMC

Hassinen A, et al. Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells. The Journal of biological chemistry. 2011;286:38329–38340. doi: 10.1074/jbc.M111.277681. PubMed DOI PMC

Axelsson MA, et al. Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins. Glycobiology. 2001;11:633–644. doi: 10.1093/glycob/11.8.633. PubMed DOI

Mukherjee S, Ghosh RN, Maxfield FR. Endocytosis. Physiological reviews. 1997;77:759–803. doi: 10.1152/physrev.1997.77.3.759. PubMed DOI

Olson LJ, Hindsgaul O, Dahms NM, Kim JJ. Structural insights into the mechanism of pH-dependent ligand binding and release by the cation-dependent mannose 6-phosphate receptor. The Journal of biological chemistry. 2008;283:10124–10134. doi: 10.1074/jbc.M708994200. PubMed DOI PMC

Wilson DW, Lewis MJ, Pelham HR. pH-dependent binding of KDEL to its receptor in vitro. The Journal of biological chemistry. 1993;268:7465–7468. PubMed

Brauer P, et al. Structural basis for pH-dependent retrieval of ER proteins from the Golgi by the KDEL receptor. Science. 2019;363:1103–1107. doi: 10.1126/science.aaw2859. PubMed DOI

Maeda Y, Ide T, Koike M, Uchiyama Y, Kinoshita T. GPHR is a novel anion channel critical for acidification and functions of the Golgi apparatus. Nature cell biology. 2008;10:1135–1145. doi: 10.1038/ncb1773. PubMed DOI

Paroutis P, Touret N, Grinstein S. The pH of the secretory pathway: measurement, determinants, and regulation. Physiology (Bethesda) 2004;19:207–215. doi: 10.1152/physiol.00005.2004. PubMed DOI

Kornak U, et al. Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2. Nat Genet. 2008;40:32–34. doi: 10.1038/ng.2007.45. PubMed DOI

Guillard M, et al. Vacuolar H+-ATPase meets glycosylation in patients with cutis laxa. Biochimica et biophysica acta. 2009;1792:903–914. doi: 10.1016/j.bbadis.2008.12.009. PubMed DOI

Rivinoja A, Pujol FM, Hassinen A, Kellokumpu S. Golgi pH, its regulation and roles in human disease. Annals of medicine. 2012;44:542–554. doi: 10.3109/07853890.2011.579150. PubMed DOI

Khayat W, et al. A recurrent missense variant in SLC9A7 causes nonsyndromic X-linked intellectual disability with alteration of Golgi acidification and aberrant glycosylation. Human molecular genetics. 2019;28:598–614. doi: 10.1093/hmg/ddy371. PubMed DOI PMC

Casey JR, Grinstein S, Orlowski J. Sensors and regulators of intracellular pH. Nature reviews. Molecular cell biology. 2010;11:50–61. doi: 10.1038/nrm2820. PubMed DOI

Braun NA, Morgan B, Dick TP, Schwappach B. The yeast CLC protein counteracts vesicular acidification during iron starvation. Journal of cell science. 2010;123:2342–2350. doi: 10.1242/jcs.068403. PubMed DOI PMC

Tarsio M, Zheng H, Smardon AM, Martinez-Munoz GA, Kane PM. Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis. The Journal of biological chemistry. 2011;286:28089–28096. doi: 10.1074/jbc.M111.251363. PubMed DOI PMC

Diakov TT, Tarsio M, Kane PM. Measurement of vacuolar and cytosolic pH in vivo in yeast cell suspensions. Journal of visualized experiments: JoVE. 2013 doi: 10.3791/50261. PubMed DOI PMC

Reifenrath M, Boles E. A superfolder variant of pH-sensitive pHluorin for in vivo pH measurements in the endoplasmic reticulum. Sci Rep. 2018;8:11985. doi: 10.1038/s41598-018-30367-z. PubMed DOI PMC

Miesenbock G, De Angelis DA, Rothman JE. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 1998;394:192–195. doi: 10.1038/28190. PubMed DOI

Rayner JC, Munro S. Identification of the MNN2 and MNN5 mannosyltransferases required for forming and extending the mannose branches of the outer chain mannans of Saccharomyces cerevisiae. The Journal of biological chemistry. 1998;273:26836–26843. doi: 10.1074/jbc.273.41.26836. PubMed DOI

Nett JH, et al. A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris. Yeast. 2011;28:237–252. doi: 10.1002/yea.1835. PubMed DOI

Renard HF, Demaegd D, Guerriat B, Morsomme P. Efficient ER exit and vacuole targeting of yeast Sna2p require two tyrosine-based sorting motifs. Traffic. 2010;11:931–946. doi: 10.1111/j.1600-0854.2010.01070.x. PubMed DOI

Mahon MJ. pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein. Adv Biosci Biotechnol. 2011;2:132–137. doi: 10.4236/abb.2011.23021. PubMed DOI PMC

Morimoto YV, Kojima S, Namba K, Minamino T. M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins. PloS one. 2011;6:e19598. doi: 10.1371/journal.pone.0019598. PubMed DOI PMC

Zimmermannova O, Salazar A, Sychrova H, Ramos J. Zygosaccharomyces rouxii Trk1 is an efficient potassium transporter providing yeast cells with high lithium tolerance. FEMS yeast research. 2015;15:fov029. doi: 10.1093/femsyr/fov029. PubMed DOI

Matsuura-Tokita K, Takeuchi M, Ichihara A, Mikuriya K, Nakano A. Live imaging of yeast Golgi cisternal maturation. Nature. 2006;441:1007–1010. doi: 10.1038/nature04737. PubMed DOI

Zhang YQ, et al. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs. PLoS pathogens. 2010;6:e1000939. doi: 10.1371/journal.ppat.1000939. PubMed DOI PMC

Kane PM. Proton Transport and pH Control in Fungi. Advances in experimental medicine and biology. 2016;892:33–68. doi: 10.1007/978-3-319-25304-6_3. PubMed DOI PMC

Martiniere A, et al. In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system. The Plant cell. 2013;25:4028–4043. doi: 10.1105/tpc.113.116897. PubMed DOI PMC

Reguera M, et al. pH Regulation by NHX-Type Antiporters Is Required for Receptor-Mediated Protein Trafficking to the Vacuole in Arabidopsis. The Plant cell. 2015;27:1200–1217. doi: 10.1105/tpc.114.135699. PubMed DOI PMC

Wu MM, et al. Organelle pH studies using targeted avidin and fluorescein-biotin. Chemistry & biology. 2000;7:197–209. doi: 10.1016/S1074-5521(00)00088-0. PubMed DOI

Lee Jong Hyun, Kim Jihoon, Park Jong‐Ho, Heo Won Do, Lee Gyun Min. Analysis of Golgi pH in Chinese hamster ovary cells using ratiometric pH‐sensitive fluorescent proteins. Biotechnology and Bioengineering. 2019;116(5):1006–1016. doi: 10.1002/bit.26920. PubMed DOI

Martinez-Munoz GA, Kane P. Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast. The Journal of biological chemistry. 2008;283:20309–20319. doi: 10.1074/jbc.M710470200. PubMed DOI PMC

Brett CL, et al. Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae. PloS one. 2011;6:e17619. doi: 10.1371/journal.pone.0017619. PubMed DOI PMC

Manolson MF, et al. The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase. The Journal of biological chemistry. 1992;267:14294–14303. PubMed

Manolson MF, et al. STV1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p. The Journal of biological chemistry. 1994;269:14064–14074. PubMed

Kawasaki-Nishi S, Bowers K, Nishi T, Forgac M, Stevens TH. The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis. The Journal of biological chemistry. 2001;276:47411–47420. doi: 10.1074/jbc.M108310200. PubMed DOI

Banerjee S, Kane PM. Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast. Molecular biology of the cell. 2017;28:2518–2530. doi: 10.1091/mbc.E17-05-0316. PubMed DOI PMC

Perzov N, Padler-Karavani V, Nelson H, Nelson N. Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis. The Journal of experimental biology. 2002;205:1209–1219. PubMed

Corbacho I, Teixido F, Olivero I, Hernandez LM. Dependence of Saccharomyces cerevisiae Golgi functions on V-ATPase activity. FEMS yeast research. 2012;12:341–350. doi: 10.1111/j.1567-1364.2011.00784.x. PubMed DOI

Stevens TH, Forgac M. Structure, function and regulation of the vacuolar (H+)-ATPase. Annu Rev Cell Dev Biol. 1997;13:779–808. doi: 10.1146/annurev.cellbio.13.1.779. PubMed DOI

Dechant R, Saad S, Ibanez AJ, Peter M. Cytosolic pH regulates cell growth through distinct GTPases, Arf1 and Gtr1, to promote Ras/PKA and TORC1 activity. Mol Cell. 2014;55:409–421. doi: 10.1016/j.molcel.2014.06.002. PubMed DOI

Wilms T, et al. The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability. PLoS genetics. 2017;13:e1006835. doi: 10.1371/journal.pgen.1006835. PubMed DOI PMC

Isom DG, et al. Coordinated regulation of intracellular pH by two glucose-sensing pathways in yeast. The Journal of biological chemistry. 2018;293:2318–2329. doi: 10.1074/jbc.RA117.000422. PubMed DOI PMC

Serrano R. In vivo glucose activation of the yeast plasma membrane ATPase. FEBS letters. 1983;156:11–14. doi: 10.1016/0014-5793(83)80237-3. PubMed DOI

Kawasaki-Nishi S, Nishi T, Forgac M. Yeast V-ATPase complexes containing different isoforms of the 100-kDa a-subunit differ in coupling efficiency and in vivo dissociation. The Journal of biological chemistry. 2001;276:17941–17948. doi: 10.1074/jbc.M010790200. PubMed DOI

Qi J, Forgac M. Cellular environment is important in controlling V-ATPase dissociation and its dependence on activity. The Journal of biological chemistry. 2007;282:24743–24751. doi: 10.1074/jbc.M700663200. PubMed DOI PMC

Finnigan GC, Hanson-Smith V, Houser BD, Park HJ, Stevens TH. The reconstructed ancestral subunit a functions as both V-ATPase isoforms Vph1p and Stv1p in Saccharomyces cerevisiae. Molecular biology of the cell. 2011;22:3176–3191. doi: 10.1091/mbc.E11-03-0244. PubMed DOI PMC

Chavez C, Bowman EJ, Reidling JC, Haw KH, Bowman BJ. Analysis of strains with mutations in six genes encoding subunits of the V-ATPase: eukaryotes differ in the composition of the V0 sector of the enzyme. The Journal of biological chemistry. 2006;281:27052–27062. doi: 10.1074/jbc.M603883200. PubMed DOI

Marshansky V. The V-ATPase a2-subunit as a putative endosomal pH-sensor. Biochemical Society transactions. 2007;35:1092–1099. doi: 10.1042/BST0351092. PubMed DOI

Baars TL, Petri S, Peters C, Mayer A. Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium. Molecular biology of the cell. 2007;18:3873–3882. doi: 10.1091/mbc.e07-03-0205. PubMed DOI PMC

Forgac M. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nature reviews. Molecular cell biology. 2007;8:917–929. doi: 10.1038/nrm2272. PubMed DOI

Maeda, Y. pH Control in Golgi Apparatus and Congenital Disorders of Glycosylation. Glycoscience: Biology and Medicine (2015).

Demaegd D, et al. Newly characterized Golgi-localized family of proteins is involved in calcium and pH homeostasis in yeast and human cells. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:6859–6864. doi: 10.1073/pnas.1219871110. PubMed DOI PMC

Snyder Nathan A., Stefan Christopher P., Soroudi Camille T., Kim Adam, Evangelista Carlos, Cunningham Kyle W. H+ and Pi Byproducts of Glycosylation Affect Ca2+ Homeostasis and Are Retrieved from the Golgi Complex by Homologs of TMEM165 and XPR1. G3: Genes|Genomes|Genetics. 2017;7(12):3913–3924. doi: 10.1534/g3.117.300339. PubMed DOI PMC

Brett CL, Tukaye DN, Mukherjee S, Rao R. The yeast endosomal Na+K+/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking. Molecular biology of the cell. 2005;16:1396–1405. doi: 10.1091/mbc.E04-11-0999. PubMed DOI PMC

Maresova L, Sychrova H. Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants. Molecular microbiology. 2005;55:588–600. doi: 10.1111/j.1365-2958.2004.04410.x. PubMed DOI

Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic acids research. 1996;24:2519–2524. doi: 10.1093/nar/24.13.2519. PubMed DOI PMC

Vida TA, Emr SD. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. The Journal of cell biology. 1995;128:779–792. doi: 10.1083/jcb.128.5.779. PubMed DOI PMC

Bolte S, Cordelieres FP. A guided tour into subcellular colocalization analysis in light microscopy. Journal of microscopy. 2006;224:213–232. doi: 10.1111/j.1365-2818.2006.01706.x. PubMed DOI

Szopinska A, Degand H, Hochstenbach JF, Nader J, Morsomme P. Rapid response of the yeast plasma membrane proteome to salt stress. Molecular & cellular proteomics: MCP. 2011;10:M111 009589. doi: 10.1074/mcp.M111.009589. PubMed DOI PMC

Colinet AS, et al. Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation. Sci Rep. 2016;6:24282. doi: 10.1038/srep24282. PubMed DOI PMC

Demaegd D, Colinet AS, Deschamps A, Morsomme P. Molecular evolution of a novel family of putative calcium transporters. PloS one. 2014;9:e100851. doi: 10.1371/journal.pone.0100851. PubMed DOI PMC