scholarly journals Demethylmenaquinone methyl transferase is a membrane domain-associated protein essential for menaquinone homeostasis in Mycobacterium smegmatis

2018 ◽  
Author(s):  
Julia Puffal ◽  
Jacob A. Mayfield ◽  
D. Branch Moody ◽  
Yasu S. Morita
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Essential Gene ◽  
Sucrose Density Gradient ◽  
Cellular Level ◽  
Distinct Region ◽  
Membrane Domain ◽  
Protein Fusion ◽  
Extra Copy

AbstractThe intracellular membrane domain (IMD) in mycobacteria is a spatially distinct region of the plasma membrane with diverse functions. Previous comparative proteomic analysis of the IMD suggested that menaquinone biosynthetic enzymes are associated with this domain. In the present study, we determined the subcellular site of these enzymes using sucrose density gradient fractionation. We found that the last two enzymes, the methyltransferase MenG, and the reductase MenJ, are associated with the IMD. MenA, the prenyltransferase that mediates the first membrane-associated step of the menaquinone biosynthesis, is associated with the conventional plasma membrane. For MenG, we additionally showed the polar enrichment of the fluorescent protein fusion colocalizing with an IMD marker protein in situ. To start dissecting the roles of IMD-associated enzymes, we further tested the physiological significance of MenG. The deletion of menG at the endogenous genomic loci was possible only when an extra copy of the gene was present, indicating that it is an essential gene in M. smegmatis. Using a tetracycline-inducible switch, we achieved gradual and partial depletion of MenG over three consecutive 24 hour subcultures. This partial MenG depletion resulted in progressive slowing of growth, which corroborated the observation that menG is an essential gene. Upon MenG depletion, there was a significant accumulation of MenG substrate, demethylmenaquinone, even though the cellular level of menaquinone, the reaction product, was unaffected. Furthermore, the growth retardation was coincided with a lower oxygen consumption rate and ATP accumulation. These results imply a previously unappreciated role of MenG in regulating menaquinone homeostasis within the complex spatial organization of mycobacterial plasma membrane.

scholarly journals Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

2022 ◽  
Author(s):  
Julia Puffal ◽  
Ian L. Sparks ◽  
James R. Brenner ◽  
Xuni Li ◽  
John D. Leszyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteomic Analysis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Membrane Domain ◽  
Protein Fusion ◽  
Final Maturation ◽  
Associated Proteins ◽  
Gradient Fractionation ◽  
Membrane Compartmentalization

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4-O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface-exposed lipid virulence factors.

scholarly journals Plant Hormones Differentially Control the Sub-Cellular Localization of Plasma Membrane Microdomains during the Early Stage of Soybean Nodulation

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1012 ◽  
Author(s):  
Zhenzhen Qiao ◽  
Prince Zogli ◽  
Marc Libault
Keyword(s):  
Plasma Membrane ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Cellular Level ◽  
Infection Thread ◽  
Membrane Microdomains ◽  
Symbiotic Interaction ◽  
Green Fluorescent ◽  
Plasma Membrane Microdomains

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and GmFWL1 or GmFLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are sufficient to trigger or inhibit the translocation of GmFWL1 and GmFLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of GmFWL1 and GmFLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation.

scholarly journals Resistance and Adaptation to Quinidine in Saccharomyces cerevisiae: Role of QDR1 (YIL120w), Encoding a Plasma Membrane Transporter of the Major Facilitator Superfamily Required for Multidrug Resistance

2001 ◽  
Vol 45 (5) ◽  
pp. 1528-1534 ◽  
Author(s):  
Patrı́cia A. Nunes ◽  
Sandra Tenreiro ◽  
Isabel Sá-Correia
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Multidrug Resistance ◽  
Fluorescent Protein ◽  
Resistance Mechanisms ◽  
Major Facilitator Superfamily ◽  
Yeast Cells ◽  
Detectable Effect ◽  
Protein Fusion ◽  
Major Facilitator

ABSTRACT As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frameYIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1.

scholarly journals Rapid removal of phagosomal ferroportin in macrophages contributes to nutritional immunity

2021 ◽  
Vol 5 (2) ◽  
pp. 459-474
Author(s):  
Ronald S. Flannagan ◽  
Tayler J. Farrell ◽  
Steven M. Trothen ◽  
Jimmy D. Dikeakos ◽  
David E. Heinrichs
Keyword(s):  
Fluorescent Protein ◽  
Critical Role ◽  
Cellular Level ◽  
Protein Fusion ◽  
Extracellular Milieu ◽  
Nutritional Immunity ◽  
Protein Receptor ◽  
Serovar Typhimurium ◽  
Green Fluorescent ◽  
Phagosomal Membrane

Abstract Nutrient sequestration is an essential facet of host innate immunity. Macrophages play a critical role in controlling iron availability through expression of the iron transport protein ferroportin (FPN), which extrudes iron from the cytoplasm to the extracellular milieu. During phagocytosis, the limiting phagosomal membrane, which derives from the plasmalemma, can be decorated with FPN and, if functional, will move iron from the cytosol into the phagosome lumen. This serves to feed iron to phagocytosed microbes and would be counterproductive to the many other known host mechanisms working to starve microbes of this essential metal. To understand how FPN is regulated during phagocytosis, we expressed FPN as a green fluorescent protein–fusion protein in macrophages and monitored its localization during uptake of various phagocytic targets, including Staphylococcus aureus, Salmonella enterica serovar Typhimurium, human erythrocytes, and immunoglobulin G opsonized latex beads. We find that FPN is rapidly removed, independently of Vps34 and PI(3)P, from early phagosomes and does not follow recycling pathways that regulate transferrin receptor recycling. Live-cell video microscopy showed that FPN movement on the phagosome is dynamic, with punctate and tubular structures forming before FPN is trafficked back to the plasmalemma. N-ethylmaleimide–sensitive factor, which disrupts soluble NSF attachment protein receptor (SNARE)–mediated membrane fusion and trafficking, prevented FPN removal from the phagosome. Our data support the hypothesis that removal of FPN from the limiting phagosomal membrane will, at the cellular level, ensure that iron cannot be pumped into phagosomes. We propose this as yet another mechanism of host nutritional immunity to subvert microbial growth.

scholarly journals Uptake of the ATP-Binding Cassette (ABC) Transporter Ste6 into the Yeast Vacuole Is Blocked in the doa4 Mutant

2001 ◽  
Vol 12 (4) ◽  
pp. 1047-1059 ◽  
Author(s):  
Sascha Losko ◽  
Frank Kopp ◽  
Andreas Kranz ◽  
Ralf Kölling
Keyword(s):  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Sucrose Density Gradient ◽  
Multivesicular Bodies ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Yeast Vacuole ◽  
Trafficking Pathway ◽  
Green Fluorescent ◽  
Gradient Fractionation

Previous experiments suggested that trafficking of thea-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells

2008 ◽  
Vol 409 (3) ◽  
pp. 731-740 ◽  
Author(s):  
Jesse Bertinato ◽  
Eleonora Swist ◽  
Louise J. Plouffe ◽  
Stephen P. J. Brooks ◽  
Mary R. L'Abbé
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Rat Tissues ◽  
Copper Uptake ◽  
Protein Fusion ◽  
Copper Transporter ◽  
African Green Monkey Kidney

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of ∼70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2–GFP (human Ctr2–green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2–GFP hyperaccumulated copper when incubated in medium supplemented with 10 μM CuSO4, whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2–GFP expression did not affect copper efflux, suggesting that hCtr2–GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2–GFP stimulated saturable copper uptake with a Km of 11.0±2.5 μM and a K0.5 of 6.9±0.7 μM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2–GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2–GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.

scholarly journals Drosophila Integrin-Linked Kinase Is Required at Sites of Integrin Adhesion to Link the Cytoskeleton to the Plasma Membrane

2001 ◽  
Vol 152 (5) ◽  
pp. 1007-1018 ◽  
Author(s):  
Christos G. Zervas ◽  
Stephen L. Gregory ◽  
Nicholas H. Brown
Keyword(s):  
Plasma Membrane ◽  
Signaling Pathways ◽  
Fluorescent Protein ◽  
Kinase Domain ◽  
Protein Fusion ◽  
Muscle Attachment ◽  
Primary Defect ◽  
Adult Wing ◽  
Integrin Linked Kinase ◽  
Green Fluorescent

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human β1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK–green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

Identification and subcellular localization of a new cystinosin isoform

2008 ◽  
Vol 294 (5) ◽  
pp. F1101-F1108 ◽  
Author(s):  
Anna Taranta ◽  
Stefania Petrini ◽  
Alessia Palma ◽  
Liliana Mannucci ◽  
Martijn J. Wilmer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Human Kidney ◽  
Nephropathic Cystinosis ◽  
Protein Fusion ◽  
Lysosomal Compartment ◽  
Renal Tubular ◽  
Green Fluorescent ◽  
Carboxy Terminal

Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last three amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of green fluorescent protein fusion plasmids in HK2 cells showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes, and in other cytosolic structures. This subcellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by [35S]cystine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells overexpressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene is not restricted to the lysosomal compartment. These finding may help elucidate the mechanisms of cell dysfunction in this disorder.

scholarly journals Tracking of Quantum Dot-labeled CFTR Shows Near Immobilization by C-Terminal PDZ Interactions

2006 ◽  
Vol 17 (12) ◽  
pp. 4937-4945 ◽  
Author(s):  
Peter M. Haggie ◽  
Jung Kyung Kim ◽  
Gergely L. Lukacs ◽  
A. S. Verkman
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Quantum Dot ◽  
Fluorescent Protein ◽  
Binding Capacity ◽  
Adaptor Protein ◽  
Single Particle Tracking ◽  
Binding Motif ◽  
Protein Fusion ◽  
Green Fluorescent

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100–200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.

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