Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation (original) (raw)
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G3 (Bethesda, Md.), 2017
Glycosylation reactions in the Golgi complex and the endoplasmic reticulum utilize nucleotide sugars as donors and produce inorganic phosphate (Pi) and acid (H+) as byproducts. Here we show that homologs of mammalian XPR1 and TMEM165 (termed Erd1 and Gdt1) recycle luminal Pi and exchange luminal H+ for cytoplasmic Ca2+, respectively, thereby promoting growth of yeast cells in low Pi and low Ca2+ environments. As expected for reversible H+/Ca2+ exchangers, Gdt1 also promoted growth in high Ca2+ environments when the Golgi-localized V-ATPase was operational but had the opposite effect when the V-ATPase was eliminated. Gdt1 activities were negatively regulated by calcineurin signaling and by Erd1, which recycled the Pi byproduct of glycosylation reactions and prevented the loss of this nutrient to the environment via exocytosis. Thus, Erd1 transports Pi in the opposite direction from XPR1 and other EXS family proteins and facilitates byproduct removal from the Golgi complex together wi...
Cell Calcium, 2015
S. cerevisiae cells respond to hypotonic stress (HTS) by a cytosolic calcium rise, either generated by an influx of calcium from extracellular medium, when calcium is available, or by a release from intracellular stores in scarcity of extracellular calcium. Calcium release from intracellular compartments is peculiarly inhibited by external calcium in a calcineurin-independent and Cch1-, but not Mid1-, driven manner. HTS-induced calcium release is also negatively regulated by the ER protein Cls2 and involves a poorly characterized protein, FLC2/YAL053W gene product, previously proposed to be required for FAD transport in the ER, albeit, due to its molecular features, it was also previously classified as an ion transporter. A computational analysis revealed that this gene and its three homologs in S. cerevisiae, together with previously identified S. pombe pkd2 and N. crassa calcium-related spray protein, belong to a fungal branch of TRP-like ion transporters related to human mucolipin and polycystin 2 calcium transporters. Moreover, disruption of FLC2 gene confers severe sensitivity to Calcofluor white and hyper-activation of the cell wall integrity MAPK cascade, suggesting a role in cell wall maintenance as previously suggested for the fission yeast homolog. Perturbation in cytosolic resting calcium concentration and hyper-activation of calcineurin in exponentially growing cells suggest a role for this transporter in calcium homeostasis in yeast. Abbreviations: [Ca 2+ ] i , intracellular calcium concentration; HACS, high affinity calcium influx system; LACS, low affinity calcium influx system; SD, synthetic medium with glucose; YPD, yeast extract peptone glucose medium; HTS, hypotonic shock.
Cell Communication and Signaling, 2018
Background: Saccharomyces cerevisiae ScGdt1 and mammalian TMEM165 are two members of the UPF0016 membrane protein family that is likely to form a new group of Ca 2+ /H + antiporter and/or a Mn 2+ transporter in the Golgi apparatus. We have previously shown that Candida albicans CaGDT1 is a functional ortholog of ScGDT1 in the response of S. cerevisiae to calcium stress. However, how CaGdt1 together with the Golgi calcium pump CaPmr1 regulate calcium homeostasis and cell wall integrity in this fungal pathogen remains unknown. Methods: Chemical sensitivity was tested by dilution assay. Cell survival was examined by measuring colony-forming units and staining with Annexin V-FITC and propidium iodide. Calcium signaling was examined by expression of downstream target gene CaUTR2, while cell wall integrity signaling was revealed by detection of phosphorylated Mkc1 and Cek1. Subcellular localization of CaGdt1 was examined through direct and indirect immunofluorescent approaches. Transcriptomic analysis was carried out with RNA sequencing. Results: This study shows that Candida albicans CaGDT1 is also a functional ortholog of ScGDT1 in the response of S. cerevisiae to cell wall stress. CaGdt1 is localized in the Golgi apparatus but at distinct sites from CaPmr1 in C. albicans. Loss of CaGDT1 increases the sensitivity of cell lacking CaPMR1 to cell wall and ER stresses. Deletion of CaGDT1 and/or CaPMR1 increases calcium uptake and activates the calcium/calcineurin signaling. Transcriptomic profiling reveals that core functions shared by CaGdt1 and CaPmr1 are involved in the regulation of cellular transport of metal ions and amino acids. However, CaGdt1 has distinct functions from CaPmr1. Chitin synthase gene CHS2 is up regulated in all three mutants, while CHS3 is only up regulated in the pmr1/pmr1 and the gdt1/gdt1 pmr1/pmr1 mutants. Five genes (DIE2, STT3, OST3, PMT1 and PMT4) of glycosylation pathway and one gene (SWI4) of the cell wall integrity (CWI) pathway are upregulated due to deletion of CaGDT1 and/or CaPMR1. Consistently, deletion of either CaPMR1 or CaGDT1 activates the CaCek1-mediated CWI signaling in a cell wall stress-independent fashion. Calcineurin function is required for the integrity of the cell wall and vacuolar compartments of cells lacking both GDT1 and CaPMR1. Conclusions: CaPmr1 is the major player in the regulation of calcium homeostasis and cell wall stress, while CaGdt1 plays a compensatory role for CaPmr1 in the Golgi compartment in C. albicans.
Unique characteristics of Ca 2+ homeostasis of the trans -Golgi compartment
Proceedings of the National Academy of Sciences, 2010
Taking advantage of a fluorescent Ca 2+ indicator selectively targeted to the trans -Golgi lumen, we here demonstrate that its Ca 2+ homeostatic mechanisms are distinct from those of the other Golgi subcompartments: ( i ) Ca 2+ uptake depends exclusively on the activity of the secretory pathway Ca 2+ ATPase1 (SPCA1), whereas the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA) is excluded; ( ii ) IP 3 generated by receptor stimulation causes Ca 2+ uptake rather than release; ( iii ) Ca 2+ release can be triggered by activation of ryanodine receptors in cells endowed with robust expression of the latter channels (e.g., in neonatal cardiac myocyte). Finally, we show that, knocking down the SPCA1, and thus altering the trans -Golgi Ca 2+ content, specific functions associated with this subcompartment, such as sorting of proteins to the plasma membrane through the secretory pathway, and the structure of the entire Golgi apparatus are dramatically altered.
Proceedings of The National Academy of Sciences, 1999
Depletion of endoplasmic reticulum Ca 2؉ stores leads to the entry of extracellular Ca 2؉ into the cytoplasm, a process termed capacitative or store-operated Ca 2؉ entry. Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca 2؉ stores were depleted by chemical and genetic means, respectively. After microinjection into Xenopus laevis oocytes, the extracts elicited a wave of increased cytoplasmic free Ca 2؉ ([Ca 2؉ ] i ) that spread from the point of injection across the oocyte. Extracts from cells with replete organellar Ca 2؉ stores were inactive. The increases depended on extracellular Ca 2؉ , were unaffected by the inositol 1,4,5-trisphosphate (IP 3 ) inhibitor heparin or an anti-IP 3 receptor antibody and were unchanged when the endoplasmic reticulum was segregated to the hemisphere opposite the injection site by centrifugation.
The FASEB Journal, 2008
In yeast, osmotic upshock causes a release of vacuolar Ca 2؉ through the mechanosensitive transient receptor potential channel, Yvc1. We screened the collection of 4810 yeast gene deletants twice for alterations in this response in an attempt to find elements that regulate the amount of vacuolar Ca 2؉ or the Yvc1 channel. Severe overresponders and underresponders to upshock were further scrutinized for their calcium content with 45 Ca and their Yvc1 electrophysiological activities under patch-clamp. The severe underresponders have lower calcium content but no change in Yvc1 activity. The strong overresponders, most of which are deleted of genes involved in cell wall metabolism, have higher calcium content. Wall mutations are known to up-regulate Ca 2؉ -calcineurin-dependent genes. It appears that stress on the cell wall induces Ca 2؉ accumulation, adaptively anticipating the need in defense or repair against future stress, including osmotic stress.-Loukin, S., Zhou, X., Kung, C., Saimi, Y. A genome-wide survey suggests an osmoprotective role for vacuolar Ca 2؉ release in cell wall-compromised yeast. FASEB J. 22, 2405-2415 (2008)
Regulation of Membrane Calcium Transport Proteins by the Surrounding Lipid Environment
Biomolecules, 2019
Calcium ions (Ca2+) are major messengers in cell signaling, impacting nearly every aspect of cellular life. Those signals are generated within a wide spatial and temporal range through a large variety of Ca2+ channels, pumps, and exchangers. More and more evidences suggest that Ca2+ exchanges are regulated by their surrounding lipid environment. In this review, we point out the technical challenges that are currently being overcome and those that still need to be defeated to analyze the Ca2+ transport protein–lipid interactions. We then provide evidences for the modulation of Ca2+ transport proteins by lipids, including cholesterol, acidic phospholipids, sphingolipids, and their metabolites. We also integrate documented mechanisms involved in the regulation of Ca2+ transport proteins by the lipid environment. Those include: (i) Direct interaction inside the protein with non-annular lipids; (ii) close interaction with the first shell of annular lipids; (iii) regulation of membrane bi...
Calcium leak from intracellular stores—the enigma of calcium signalling
Cell Calcium, 2002
Wherever you travel through the cytoplasm of the cells you will find organelles with internal [Ca 2+ ] levels higher than in the surrounding cytosol. This is particularly true of the endoplasmic reticulum (ER) (or sarcoplasmic reticulum (SR) in muscle cells); such organelles serve as the main sources of releasable Ca 2+ for cytosolic cellular signalling. Calcium pumps of the SERCA family (sarcoplasmic and endoplasmic reticulum calcium ATP-ases) import calcium into the organelle lumen. The other mechanism that is responsible for the steady state calcium level within the lumen of ER or SR is a calcium leak that balances the influx created by the pumps. The leak remains the most enigmatic of the processes involved in calcium regulation. The molecular nature of the leak mechanism is not known. The basal leak is a relatively slow process, which is difficult to investigate and which is easily outmatched (both in the amplitude of calcium responses and in attractiveness to experimenters) by substantially faster second messenger-induced release. Nevertheless, information on the properties of the calcium leak, although thinly scattered through the pages of PubMed, has been slowly accumulating. In this review we will discuss the properties of the calcium leak and speculate about possible mechanisms, which could mediate this process.