scholarly journals Drs2p-related P-type ATPases Dnf1p and Dnf2p Are Required for Phospholipid Translocation across the Yeast Plasma Membrane and Serve a Role in Endocytosis

2003 ◽  
Vol 14 (3) ◽  
pp. 1240-1254 ◽  
Author(s):  
Thomas Pomorski ◽  
Ruben Lombardi ◽  
Howard Riezman ◽  
Philippe F. Devaux ◽  
Gerrit van Meer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Structural Similarity ◽  
Trinitrobenzene Sulfonic Acid ◽  
Yeast Plasma Membrane ◽  
Outer Leaflet ◽  
Sphingolipid Analogs ◽  
Dependent Transport ◽  
P Type ◽  
Atpase Ii

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by ATP-driven lipid transporters whose identities are unknown. The yeast plasma membrane contains two P-type ATPases, Dnf1p and Dnf2p, with structural similarity to ATPase II, a candidate aminophospholipid translocase from bovine chromaffin granules. Loss of Dnf1p and Dnf2p virtually abolished ATP-dependent transport of NBD-labeled phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine from the outer to the inner plasma membrane leaflet, leaving transport of sphingolipid analogs unaffected. Labeling with trinitrobenzene sulfonic acid revealed that the amount of phosphatidylethanolamine exposed on the surface of Δdnf1Δdnf2 cells increased twofold relative to wild-type cells. Phosphatidylethanolamine exposure by Δdnf1Δdnf2 cells further increased upon removal of Drs2p, an ATPase II homolog in the yeast Golgi. These changes in lipid topology were accompanied by a cold-sensitive defect in the uptake of markers for bulk-phase and receptor-mediated endocytosis. Our findings demonstrate a requirement for Dnf1p and Dnf2p in lipid translocation across the yeast plasma membrane. Moreover, it appears that Dnf1p, Dnf2p and Drs2p each help regulate the transbilayer lipid arrangement in the plasma membrane, and that this regulation is critical for budding endocytic vesicles.

scholarly journals Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists

1997 ◽  
Vol 322 (3) ◽  
pp. 823-828 ◽  
Author(s):  
Irma ROMERO ◽  
Ana M. MALDONADO ◽  
Pilar ERASO
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Glucose Regulation ◽  
Wild Type ◽  
Calmodulin Antagonists ◽  
Dependent Protein Kinase ◽  
Yeast Plasma Membrane ◽  
Dependent Protein

Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitroeffect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of ≈1 μM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

scholarly journals The tertiary structure of the human Xkr8–Basigin complex that scrambles phospholipids at plasma membranes

2021 ◽  
Vol 28 (10) ◽  
pp. 825-834
Author(s):  
Takaharu Sakuragi ◽  
Ryuta Kanai ◽  
Akihisa Tsutsumi ◽  
Hirotaka Narita ◽  
Eriko Onishi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Tertiary Structure ◽  
Vital Role ◽  
Tryptophan Residue ◽  
Head Group ◽  
Salt Bridges ◽  
Phospholipid Scramblase ◽  
Outer Leaflet

AbstractXkr8–Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8–Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8–Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.

Vacuolar-type H(+)-ATPases are functionally expressed in plasma membranes of human tumor cells

1993 ◽  
Vol 265 (4) ◽  
pp. C1015-C1029 ◽  
Author(s):  
R. Martinez-Zaguilan ◽  
R. M. Lynch ◽  
G. M. Martinez ◽  
R. J. Gillies
Keyword(s):  
Plasma Membrane ◽  
Tumor Cells ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Human Tumor ◽  
Atp Depletion ◽  
Second Phase ◽  
Bafilomycin A1 ◽  
P Type

Mammalian cells generally regulate their intracellular pH (pHi) via collaboration between Na(+)-H+ exchanger and HCO3- transport. In addition, a number of normal mammalian cells have been identified that express H(+)-adenosinetriphosphatases (ATPases) in their plasma membranes. Because tumor cells often maintain a high pHi, we hypothesized that they might functionally express H(+)-ATPases in their plasma membranes. In the first phase of the present study, we screened 19 normal and tumorigenic human cell lines for the presence of plasmalemmal H(+)-ATPase activity using bafilomycin A1 to inhibit V-type H(+)-ATPase and Sch-28080 to inhibit P-type H(+)-K(+)-ATPase. Bafilomycin A1 decreased pHi in the six tumor cell lines with the highest resting pHi in the absence of HCO3-. Sch-28080 did not affect pHi in any of the human cells. Simultaneous measurement of pH in the cytoplasm and in the endosomes/lysosomes localized the activity of bafilomycin to the plasma membrane in three cell lines. In the second phase of this study, these three cell lines were shown to recover from NH4(+)-induced acid loads in the absence of Na+. This recovery was inhibited by N-ethylmaleimide, bafilomycin A1, and ATP depletion and was not significantly affected by vanadate, Sch-28080, or hexamethyl amiloride. These results indicate that a vacuolar type H(+)-ATPase is expressed in the plasma membrane of some tumor cells.

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.

scholarly journals Diffusion Coefficient of Fluorescent Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane of Cells

2008 ◽  
Vol 19 (4) ◽  
pp. 1663-1669 ◽  
Author(s):  
Urszula Golebiewska ◽  
Marian Nyako ◽  
William Woturski ◽  
Irina Zaitseva ◽  
Stuart McLaughlin
Keyword(s):  
Diffusion Coefficient ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Fluorescence Correlation Spectroscopy ◽  
Plasma Membranes ◽  
Diffusion Constant ◽  
Cell Types ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Outer Leaflet

Phosphatidylinositol 4,5-bisphosphate (PIP2) controls a surprisingly large number of processes in cells. Thus, many investigators have suggested that there might be different pools of PIP2 on the inner leaflet of the plasma membrane. If a significant fraction of PIP2 is bound electrostatically to unstructured clusters of basic residues on membrane proteins, the PIP2 diffusion constant, D, should be reduced. We microinjected micelles of Bodipy TMR-PIP2 into cells, and we measured D on the inner leaflet of fibroblasts and epithelial cells by using fluorescence correlation spectroscopy. The average ± SD value from all cell types was D = 0.8 ± 0.2 μm2/s (n = 218; 25°C). This is threefold lower than the D in blebs formed on Rat1 cells, D = 2.5 ± 0.8 μm2/s (n = 26). It is also significantly lower than the D in the outer leaflet or in giant unilamellar vesicles and the diffusion coefficient for other lipids on the inner leaflet of these cell membranes. The simplest interpretation is that approximately two thirds of the PIP2 on inner leaflet of these plasma membranes is bound reversibly.

Biogenesis and function of the yeast plasma-membrane H(+)-ATPase

2000 ◽  
Vol 203 (1) ◽  
pp. 155-160 ◽  
Author(s):  
A. Ambesi ◽  
M. Miranda ◽  
V.V. Petrov ◽  
C.W. Slayman
Keyword(s):  
Plasma Membrane ◽  
Atp Hydrolysis ◽  
Proton Pumping ◽  
Site Directed Mutagenesis ◽  
Animal Cells ◽  
Alpha Helices ◽  
Yeast Plasma Membrane ◽  
High Affinity ◽  
And Function ◽  
P Type

One of the most abundant proteins in the yeast plasma membrane is the P-type H(+)-ATPase that pumps protons out of the cell, supplying the driving force for a wide array of H(+)-dependent cotransporters. The ATPase is a 100 kDa polypeptide, anchored in the lipid bilayer by 10 transmembrane alpha-helices. It is structurally and functionally related to the P-type Na(+),K(+)-, H(+),K(+)- and Ca(2+)-ATPases of animal cells and the H(+)-ATPases of plant cells, and it shares with them a characteristic reaction mechanism in which ATP is split to ADP and inorganic phosphate (P(i)) via a covalent beta-aspartyl phosphate intermediate. Cryoelectron microscopic images of the H(+)-ATPase of Neurospora crassa and the sarcoplasmic reticulum Ca(2+)-ATPase of animal cells have recently been obtained at 8 nm resolution. The membrane-embedded portion of the molecule, which presumably houses the cation translocation pathway, is seen to be connected via a narrow stalk to a large, multidomained cytoplasmic portion, known to contain the ATP-binding and phosphorylation sites. In parallel with the structural studies, efforts are being made to dissect structure/function relationships in several P-type ATPases by means of site-directed mutagenesis. This paper reviews three phenotypically distinct classes of mutant that have resulted from work on the yeast PMA1 H(+)-ATPase: (1) mutant ATPases that are poorly folded and retained in the endoplasmic reticulum; (2) mutants in which the conformational equilibrium has been shifted from the E(2) state, characterized by high affinity for vanadate, to the E(1) state, characterized by high affinity for ATP; and (3) mutants with altered coupling between ATP hydrolysis and proton pumping. Although much remains to be learned before the transport mechanism can be fully understood, these mutants serve to identify critical parts of the polypeptide that are required for protein folding, conformational change and H(+):ATP coupling.

scholarly journals Plasma membrane aminoglycerolipid flippase function is required for signaling competence in the yeast mating pheromone response pathway

2015 ◽  
Vol 26 (1) ◽  
pp. 134-150 ◽  
Author(s):  
Elodie Sartorel ◽  
Evelyne Barrey ◽  
Rebecca K. Lau ◽  
Jeremy Thorner
Keyword(s):  
Plasma Membrane ◽  
Triple Mutant ◽  
Membrane Asymmetry ◽  
Outer Leaflet ◽  
Pheromone Signaling ◽  
Elevated Rate ◽  
And Function ◽  
Phosphatidylinositol 4 ◽  
P Type ◽  
Pronounced Reduction

The class 4 P-type ATPases (“flippases”) maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MATa cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection (“shmoo”) tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MATa dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2⋅Cdc28-initiated degradation. Similarly, a MATa dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

Microtubule Dynamics and Organelle Transport in Reticulomyxa

1990 ◽  
Vol 48 (3) ◽  
pp. 194-195
Author(s):  
Yih-Tai Chen ◽  
Ursula Euteneuer ◽  
Ken B. Johnson ◽  
Michael P. Koonce ◽  
Manfred Schliwa
Keyword(s):  
Plasma Membrane ◽  
Light Microscopy ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Microtubule Dynamics ◽  
Model Systems ◽  
Organelle Transport ◽  
Biochemical Characteristics ◽  
Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

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