scholarly journals Targeting the fungal plasma membrane proton pump.

1995 ◽  
Vol 42 (4) ◽  
pp. 481-496 ◽  
Author(s):  
B C Monk ◽  
A B Mason ◽  
T B Kardos ◽  
D S Perlin
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Proton Pump ◽  
Fungal Pathogens ◽  
Mutational Analysis ◽  
Proton Pumping ◽  
Proton Pumps ◽  
Essential Enzyme ◽  
Altered Cell ◽  
P Type

The need for new mechanistic classes of broad spectrum antifungal agents has prompted development of the membrane sector and ectodomain of the plasma membrane proton pumping ATPase as an antifungal target. The fungal proton pump is a highly abundant, essential enzyme in Saccharomyces cerevisiae. It belongs to the family of P-type ATPases, a class of enzymes that includes the Na+,K(+)-ATPase and the gastric H+,K(+)-ATPase. These enzymes are cell surface therapeutic targets for the cardiac glycosides and several anti-ulcer drugs, respectively. The effects of acid-activated omeprazole show that extensive inhibition of the S. cerevisiae ATPase is fungicidal. Fungal proton pumps possess elements within their transmembrane loops that distinguish them from other P-type ATPases. These loops, such as the conformationally sensitive transmembrane loop 1+2, can attenuate the activity of the enzyme. Expression in S. cerevisiae of fully functional chimeric ATPases that contain a foreign target comprising transmembrane loops 1+2 and/or 3+4 from the fungal pathogen Candida albicans suggests that these loops operate as a domain. The chimera containing C. albicans transmembrane loops 1+2 and 3+4 provides a prototype for mutational analysis of the target region and the screening of inhibitors directed against opportunistic fungal pathogens. Panels of mutants with modified ATPase regulation or with altered cell surface cysteine residues are also described. Information about the ATPase membrane sector and ectodomain has been integrated into a model of this region.

scholarly journals Eisosomes are metabolically regulated storage compartments for APC-type nutrient transporters

2018 ◽  
Vol 29 (17) ◽  
pp. 2113-2127 ◽  
Author(s):  
Akshay Moharir ◽  
Lincoln Gay ◽  
Daniel Appadurai ◽  
James Keener ◽  
Markus Babst
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Proton Pumping ◽  
Proton Gradient ◽  
Lipid Domains ◽  
Nutrient Transporters ◽  
Major Function ◽  
Show Evidence ◽  
Higher Eukaryotes ◽  
Pumping Activity

Eisosomes are lipid domains of the yeast plasma membrane that share similarities to caveolae of higher eukaryotes. Eisosomes harbor APC-type nutrient transporters for reasons that are poorly understood. Our analyses support the model that eisosomes function as storage compartments, keeping APC transporters in a stable, inactive state. By regulating eisosomes, yeast is able to balance the number of proton-driven APC transporters with the proton-pumping activity of Pma1, thereby maintaining the plasma membrane proton gradient. Environmental or metabolic changes that disrupt the proton gradient cause the rapid restructuring of eisosomes and results in the removal of the APC transporters from the cell surface. Furthermore, we show evidence that eisosomes require the presence of APC transporters, suggesting that regulating activity of nutrient transporters is a major function of eisosomes.

scholarly journals Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase.

1992 ◽  
Vol 119 (1) ◽  
pp. 111-122 ◽  
Author(s):  
I Sabolic ◽  
F Wuarin ◽  
L B Shi ◽  
A S Verkman ◽  
D A Ausiello ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proton Pump ◽  
Transmembrane Domain ◽  
Collecting Duct ◽  
Water Channels ◽  
Proton Pumping ◽  
Apical Plasma Membrane ◽  
Principal Cells ◽  
Kidney Collecting Duct ◽  
Early Endosomes

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.

scholarly journals Role for a P-type H+-ATPase in the acidification of the endocytic pathway of Trypanosoma cruzi

2005 ◽  
Vol 392 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Mauricio Vieira ◽  
Peter Rohloff ◽  
Shuhong Luo ◽  
Narcisa L. Cunha-E-Silva ◽  
Wanderley De Souza ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Subcellular Fractionation ◽  
Etiologic Agent ◽  
Proton Pumping ◽  
Endocytic Pathway ◽  
Immunogold Electron Microscopy ◽  
Intermediate Form ◽  
Confocal Immunofluorescence Microscopy ◽  
P Type

Previous studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, have resulted in the cloning and sequencing of a pair of tandemly linked genes (TcHA1 and TcHA2) that encode P (phospho-intermediate form)-type H+-ATPases with homology to fungal and plant proton-pumping ATPases. In the present study, we demonstrate that these pumps are present in the plasma membrane and intracellular compartments of three different stages of T. cruzi. The main intracellular compartment containing these ATPases in epimastigotes was identified as the reservosome. This identification was achieved by immunofluorescence assays and immunoelectron microscopy showing their co-localization with cruzipain, and by subcellular fractionation and detection of their activity. ATP-dependent proton transport by isolated reservosomes was sensitive to vanadate and insensitive to bafilomycin A1, which is in agreement with the localization of P-type H+-ATPases in these organelles. Analysis by confocal immunofluorescence microscopy revealed that epitope–tagged TcHA1-Ty1 and TcHA2-Ty1 gene products are localized in the reservosomes, whereas the TcHA1-Ty1 gene product is additionally present in the plasma membrane. Immunogold electron microscopy showed the presence of the H+-ATPases in other compartments of the endocytic pathway such as the cytostome and endosomal vesicles, suggesting that in contrast with most cells investigated until now, the endocytic pathway of T. cruzi is acidified by a P-type H+-ATPase.

scholarly journals In vivo cross-linking supports a head-to-tail mechanism for regulation of the plant plasma membrane P-type H+-ATPase

2018 ◽  
Vol 293 (44) ◽  
pp. 17095-17106 ◽  
Author(s):  
Thao T. Nguyen ◽  
Grzegorz Sabat ◽  
Michael R. Sussman
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Unnatural Amino Acid ◽  
Proton Pumping ◽  
Intramolecular Interactions ◽  
Cross Linking ◽  
Regulatory Domain ◽  
P Type ◽  
Plant Plasma Membrane

In higher plants, a P-type proton-pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved, but direct chemical evidence for which ones are specifically interacting with the C terminus is lacking. In this study, we used in vivo cross-linking experiments with a photoreactive unnatural amino acid, p-benzoylphenylalanine, and tandem MS to obtain direct evidence that the C-terminal regulatory domain interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a “head-to-tail” organization of ATPase monomers in which the C-terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and it is consistent with prior studies suggesting that within the membrane the protein can be organized as homopolymers, including dimers, trimers, and hexamers.

scholarly journals Loss of P4 ATPases Drs2p and Dnf3p Disrupts Aminophospholipid Transport and Asymmetry in Yeast Post-Golgi Secretory Vesicles

2006 ◽  
Vol 17 (4) ◽  
pp. 1632-1642 ◽  
Author(s):  
Nele Alder-Baerens ◽  
Quirine Lisman ◽  
Lambert Luong ◽  
Thomas Pomorski ◽  
Joost C.M. Holthuis
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Budding Yeast ◽  
Secretory Vesicles ◽  
Trans Golgi Network ◽  
P Type ◽  
Golgi Network

Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.

Mutational analysis of the carboxy-terminal phosphorylation site of GLUT-4 in 3T3-L1 adipocytes

1998 ◽  
Vol 275 (3) ◽  
pp. E412-E422 ◽  
Author(s):  
Brad J. Marsh ◽  
Sally Martin ◽  
Derek R. Melvin ◽  
Laura B. Martin ◽  
Richard A. Alm ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Glut 4 ◽  
Insulin Dependent ◽  
Glut 4 Translocation ◽  
Intracellular Vesicles ◽  
Carboxy Terminal ◽  
Golgi Network

The carboxy terminus of GLUT-4 contains a functional internalization motif (Leu-489Leu-490) that helps maintain its intracellular distribution in basal adipocytes. This motif is flanked by the major phosphorylation site in this protein (Ser-488), which may play a role in regulating GLUT-4 trafficking in adipocytes. In the present study, the targeting of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has been examined in stably transfected 3T3-L1 adipocytes. The trafficking of SAG was not significantly different from that of GLUT-4 in several respects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surface and was enriched in small intracellular vesicles. Second, SAG exhibited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its phosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containing the Golgi-derived γ-adaptin subunit of AP-1 and that this localization is enhanced when Ser-488 is mutated to alanine. We conclude that the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may play a role in intracellular sorting at the trans-Golgi network but does not play a major role in the regulated movement of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes.

scholarly journals The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants

2020 ◽  
Vol 295 (44) ◽  
pp. 14793-14804 ◽  
Author(s):  
Jessica E. Besaw ◽  
Wei-Lin Ou ◽  
Takefumi Morizumi ◽  
Bryan T. Eger ◽  
Juan D. Sanchez Vasquez ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Crystal Structures ◽  
Proton Pump ◽  
Chloride Transport ◽  
Chloride Ion ◽  
Proton Pumping ◽  
Single Amino Acid ◽  
Target Cells ◽  
Proton Pumps ◽  
Transport Pathway

Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.

scholarly journals Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola

2015 ◽  
Vol 197 (16) ◽  
pp. 2704-2712 ◽  
Author(s):  
J. L. Keffer ◽  
M. W. Hahn ◽  
J. A. Maresca
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
High Performance ◽  
Proton Pumping ◽  
Host Cells ◽  
Metagenomic Data ◽  
Proton Pumps ◽  
Content Type ◽  
Knowing That ◽  
Light Capture

ABSTRACTRhodopsin-encoding microorganisms are common in many environments. However, knowing that rhodopsin genes are present provides little insight into how the host cells utilize light. The genome of the freshwater actinobacteriumRhodoluna lacicolaencodes a rhodopsin of the uncharacterized actinorhodopsin family. We hypothesized that actinorhodopsin was a light-activated proton pump and confirmed this by heterologously expressingR. lacicolaactinorhodopsin in retinal-producingEscherichia coli. However, cultures ofR. lacicoladid not pump protons, even though actinorhodopsin mRNA and protein were both detected. Proton pumping inR. lacicolawas induced by providing exogenous retinal, suggesting that the cells lacked the retinal cofactor. We used high-performance liquid chromatography (HPLC) and oxidation of accessory pigments to confirm thatR. lacicoladoes not synthesize retinal. These results suggest that in some organisms, the actinorhodopsin gene is constitutively expressed, but rhodopsin-based light capture may require cofactors obtained from the environment.IMPORTANCEUp to 70% of microbial genomes in some environments are predicted to encode rhodopsins. Because most microbial rhodopsins are light-activated proton pumps, the prevalence of this gene suggests that in some environments, most microorganisms respond to or utilize light energy. Actinorhodopsins were discovered in an analysis of freshwater metagenomic data and subsequently identified in freshwater actinobacterial cultures. We hypothesized that actinorhodopsin from the freshwater actinobacteriumRhodoluna lacicolawas a light-activated proton pump and confirmed this by expressing actinorhodopsin in retinal-producingEscherichia coli. Proton pumping inR. lacicolawas induced only after both light and retinal were provided, suggesting that the cells lacked the retinal cofactor. These results indicate that photoheterotrophy in this organism and others may require cofactors obtained from the environment.

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