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ACS Chemical Biology Jan 2022Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles...
Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)-Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.
Topics: Adenosine Triphosphatases; Brefeldin A; Endoplasmic Reticulum; Golgi Apparatus; Humans; Hydrogen-Ion Concentration; Macrolides; Microscopy, Fluorescence; Optical Imaging; Protein Transport; Secretory Pathway
PubMed: 35000377
DOI: 10.1021/acschembio.1c00907 -
Angewandte Chemie (International Ed. in... Mar 2015The important biochemical probe molecule brefeldin A (1) has served as an inspirational target in the past, but none of the many routes has actually delivered more...
The important biochemical probe molecule brefeldin A (1) has served as an inspirational target in the past, but none of the many routes has actually delivered more than just a few milligrams of product, where documented. The approach described herein is clearly more efficient; it hinges upon the first implementation of ruthenium-catalyzed trans-hydrogenation in natural products total synthesis. Because this unorthodox reaction is selective for the triple bond and does not touch the transannular alkene or the lactone site of the cycloalkyne, it outperforms the classical Birch-type reduction that could not be applied at such a late stage. Other key steps en route to 1 comprise an iron-catalyzed reductive formation of a non-terminal alkyne, an asymmetric propiolate carbonyl addition mediated by a bulky amino alcohol, and a macrocyclization by ring-closing alkyne metathesis catalyzed by a molybdenum alkylidyne.
Topics: Alkenes; Brefeldin A; Catalysis; Cyclization; Hydrogenation; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Molybdenum; Oxidation-Reduction; Ruthenium Compounds
PubMed: 25651519
DOI: 10.1002/anie.201411618 -
Journal of Virology Jun 2021Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the endoplasmic reticulum (ER)...
Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the endoplasmic reticulum (ER) membrane into a functional N-terminal signal peptide (SP) and the C terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, and yet it was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug brefeldin A (BFA), which interferes with the ER-to-Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels that were not rescued by proteasomal or lysosomal inhibitors. A Rem mutant lacking glycosylation was cleaved into SP and Rem-CT but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not reach this BFA-dependent compartment. Treatment with endoglycosidase H indicated that Rem-CT does not traffic through the Golgi apparatus. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, was colocalized with Rab5-positive (Rab5) early endosomes. The expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (containing a mutation of threonine to asparagine at position 31 [T31N]) mimicked the effects of BFA by reducing Rem-CT levels and increased Rem-CT association with early and late endosomes. Inhibition of the AAA ATPase p97/VCP rescued Rem-CT in the presence of BFA or DN Arf1 and prevented localization to Rab5 endosomes. Thus, Rem-CT uses an unconventional p97-mediated scheme for trafficking to early endosomes. Mouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP uses the p97/VCP ATPase to elude ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used mutants, inhibitors, and confocal microscopy to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation and the p97 ATPase for early endosome trafficking without passage through the Golgi apparatus. Thus, Rem-CT uses a novel intracellular trafficking pathway, potentially impacting host antiviral immunity.
Topics: Adenosine Triphosphatases; Biological Transport; Brefeldin A; Endoplasmic Reticulum; Endosomes; HEK293 Cells; Humans; Mammary Tumor Virus, Mouse; Microscopy, Confocal; Nuclear Proteins; Protein Precursors; Viral Envelope Proteins
PubMed: 33952644
DOI: 10.1128/JVI.00531-21 -
Journal of Cell Science Jul 2017Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether...
Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.
Topics: A549 Cells; ADP-Ribosylation Factor 1; Anti-Bacterial Agents; Antigens, Neoplasm; Autophagy; Brefeldin A; Dysentery, Bacillary; Gene Knockdown Techniques; Golgi Apparatus; Guanine Nucleotide Exchange Factors; HEK293 Cells; HT29 Cells; HeLa Cells; Humans; Shigella flexneri; Vesicular Transport Proteins
PubMed: 28536105
DOI: 10.1242/jcs.199521 -
The Journal of Biological Chemistry Oct 1996In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated...
In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus. Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and beta-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport. Employing cell lines possessing inducible forms of ARF, we demonstrate here a tight coupling of the nucleotide-bound state of ARF and the subcellular localization of Cdc42Hs. Specifically, the expression of wild-type ARF had no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and beta-COP, expression of a GTPase-deficient form of ARF (ARF(Q71L)) renders these Golgi-localized proteins resistant to brefeldin A treatment (; ). Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide resulted in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. These results suggest that Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane.
Topics: Animals; Brefeldin A; Cell Cycle Proteins; Cyclopentanes; GTP-Binding Proteins; Golgi Apparatus; Microscopy, Fluorescence; Rabbits; cdc42 GTP-Binding Protein
PubMed: 8900167
DOI: 10.1074/jbc.271.43.26850 -
Proceedings of the National Academy of... Mar 2004Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an approximately 200-kDa brefeldin A-inhibited guanine nucleotide-exchange protein that...
Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an approximately 200-kDa brefeldin A-inhibited guanine nucleotide-exchange protein that preferentially activates ADP-ribosylation factor 1 (ARF1) and ARF3. BIG1 was found in cytosol in a multiprotein complex with a similar ARF-activating protein, BIG2, which is also an A kinase-anchoring protein. In HepG2 cells growing with serum, BIG1 was primarily cytosolic and Golgi-associated. After incubation overnight without serum, a large fraction of endogenous BIG1 was in the nuclei. By confocal immunofluorescence microscopy, BIG1 was localized with nucleoporin p62 at the nuclear envelope (probably during nucleocytoplasmic transport) and also in nucleoli, clearly visible against the less concentrated overall matrix staining. BIG1 was also identified by Western blot analyses in purified subnuclear fractions (e.g., nucleoli and nuclear matrix). Antibodies against BIG1, nucleoporin, or nucleolin coimmunoprecipitated the other two proteins from purified nuclei. In contrast, BIG2 was not associated with nuclear BIG1. Also of note, ARF was never detected among proteins precipitated from purified nuclei by anti-BIG1 antibodies, although microscopically the two proteins do appear sometimes to be colocalized in the nucleus. These data are consistent with independent intracellular movements and actions of BIG1 and BIG2, and they are also evidence of the participation of BIG1 in both Golgi and nuclear functions.
Topics: Adenosine Diphosphate Ribose; Brefeldin A; Cell Line, Tumor; Cell Nucleolus; Culture Media; GTP-Binding Proteins; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Humans; Liver Neoplasms; Microscopy, Confocal; Microscopy, Fluorescence; Protein Transport; Tacrolimus
PubMed: 14973189
DOI: 10.1073/pnas.0307345101 -
The Plant Journal : For Cell and... Nov 2022Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) is a glycolytic enzyme, but undergoes stress-induced nuclear translocation for moonlighting. We previously...
Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) is a glycolytic enzyme, but undergoes stress-induced nuclear translocation for moonlighting. We previously reported that in response to heat stress, GAPC accumulated in the nucleus to modulate transcription and thermotolerance. Here we show a cellular and molecular mechanism that mediates heat-induced nuclear translocation of cytosolic GAPC in Arabidopsis thaliana. Heat-induced GAPC nuclear accumulation and plant heat tolerance were reduced in Arabidopsis phospholipase D (PLD) knockout mutants of pldδ and pldα1pldδ, but not of pldα1. These changes were restored to wild type by genetic complementation with active PLDδ, but not with catalytically inactive PLDδ. GAPC overexpression enhanced the seedling thermotolerance and the expression of heat-inducible genes, but this effect was abolished in the pldδ background. Heat stress elevated the levels of the PLD product phosphatidic acid (PA) in the nucleus in wild type, but not in pldδ plants. Lipid labeling demonstrated the heat-induced nuclear co-localization of PA and GAPC, which was impaired by zinc, which inhibited the PA-GAPC interaction, and by the membrane trafficking inhibitor brefeldin A (BFA). The GAPC nuclear accumulation and seedling thermotolerance were also decreased by treatment with zinc or BFA. Our data suggest that PLDδ and PA are critical for the heat-induced nuclear translocation of GAPC. We propose that PLDδ-produced PA mediates the process via lipid-protein interaction and that the lipid mediation acts as a cellular conduit linking stress perturbations at cell membranes to nuclear functions in plants coping with heat stress.
Topics: Arabidopsis; Phosphatidic Acids; Arabidopsis Proteins; Phospholipases; Phospholipase D; Glyceraldehyde-3-Phosphate Dehydrogenases; Seedlings; Brefeldin A; Zinc
PubMed: 36111506
DOI: 10.1111/tpj.15981 -
The Plant Journal : For Cell and... Jul 2021The xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in cell wall assembly and growth regulation, cleaving and re-joining hemicellulose chains in...
The xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in cell wall assembly and growth regulation, cleaving and re-joining hemicellulose chains in the xyloglucan-cellulose network. Here, in a homologous system, we compare the secretion patterns of XTH11, XTH33 and XTH29, three members of the Arabidopsis thaliana XTH family, selected for the presence (XTH11 and XTH33) or absence (XTH29) of a signal peptide, and the presence of a transmembrane domain (XTH33). We show that XTH11 and XTH33 reached, respectively, the cell wall and plasma membrane through a conventional protein secretion (CPS) pathway, whereas XTH29 moves towards the apoplast following an unconventional protein secretion (UPS) mediated by exocyst-positive organelles (EXPOs). All XTHs share a common C-terminal functional domain (XET-C) that, for XTH29 and a restricted number of other XTHs (27, 28 and 30), continues with an extraterminal region (ETR) of 45 amino acids. We suggest that this region is necessary for the correct cell wall targeting of XTH29, as the ETR-truncated protein never reaches its final destination and is not recruited by EXPOs. Furthermore, quantitative real-time polymerase chain reaction analyses performed on 4-week-old Arabidopsis seedlings exposed to drought and heat stress suggest a different involvement of the three XTHs in cell wall remodeling under abiotic stress, evidencing stress-, organ- and time-dependent variations in the expression levels. Significantly, XTH29, codifying the only XTH that follows a UPS pathway, is highly upregulated with respect to XTH11 and XTH33, which code for CPS-secreted proteins.
Topics: Arabidopsis; Arabidopsis Proteins; Brefeldin A; Cell Membrane; Cell Wall; Dehydration; Glycosyltransferases; Golgi Apparatus; Heat-Shock Response; Protein Translocation Systems
PubMed: 33932060
DOI: 10.1111/tpj.15301 -
FEBS Letters Oct 2019CREB3 is a transcription factor localized to the ER. Here, we investigated endogenous CREB3 expression in HEK293 cells using pharmacological and genome editing...
CREB3 is a transcription factor localized to the ER. Here, we investigated endogenous CREB3 expression in HEK293 cells using pharmacological and genome editing approaches. Full-length CREB3 detected under resting conditions disappeared following treatment with tunicamycin, brefeldin A and nigericin. Treatment with cycloheximide and MG132 indicated that endogenous CREB3 is a proteasome substrate. Using cells deficient for the ER-associated protein degradation (ERAD) factors SEL1L and Herp, we demonstrate that SEL1L, but not Herp, plays a crucial role in the posttranslational regulation of full-length CREB3 expression. In addition, kifunensine, an α-mannosidase inhibitor, remarkably increased full-length CREB3 expression. Our study suggests that endogenous full-length CREB3 is a novel substrate for ERAD and identifies unique cellular signals distinct from those in canonical ER stress.
Topics: Brefeldin A; Cyclic AMP Response Element-Binding Protein; Cycloheximide; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; HEK293 Cells; Humans; Leupeptins; Membrane Proteins; Nigericin; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Proteins; Tunicamycin
PubMed: 31291699
DOI: 10.1002/1873-3468.13535 -
Cell Apr 1999
Review
Topics: ADP-Ribosylation Factors; Animals; Brefeldin A; GTP-Binding Proteins; Golgi Apparatus; Guanosine Diphosphate; Models, Molecular; Mutation
PubMed: 10219235
DOI: 10.1016/s0092-8674(00)80724-2