Transbilayer Movement of Fluorescent Phospholipids in <i>Bacillus megaterium</i> Membrane Vesicles

Hrafnsdóttir, Sigrún; Nichols, J. Wylie; Menon, Anant K.

doi:10.1021/bi962513h

Cited by 61 publications

(66 citation statements)

References 31 publications

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“…To ensure stable membrane growth, energy-independent flippases mediate rapid, bidirectional, and rather unspecific phospholipid flip-flop with half-times of minutes or less (4 -7). A similar flippase activity was also found in the bacterial inner membrane, where lipid synthesis occurs likewise at the cytoplasmic leaflet (8).…”

supporting

confidence: 67%

Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

Papadopulos

Vehring

López‐Montero

et al. 2007

Journal of Biological Chemistry

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Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.In lipid bilayers the spontaneous movement of major phospholipids, e.g. of phosphatidylcholine (PC), 6 between the two monolayers is slow, with half-times on the order of hours or even days. However, lipid topology of cellular membranes results from a continuous bidirectional movement (flip-flop) of lipids between the two leaflets in which specific membrane proteins, so called flippases, play an essential role (1, 2). Energyindependent flippases allow phospholipids to equilibrate rapidly between the two monolayers, whereas energy-dependent flippases mediate a net transfer of specific phospholipids to one leaflet of the membrane. Candidates for the latter flippase are members of a conserved subfamily of P-type ATPases (2) as well as ATP binding cassette transporters (3).In eukaryotes the cytoplasmic leaflet of the endoplasmic reticulum (ER) membrane is the major site of phospholipid biosynthesis. To ensure stable membrane growth, energy-independent flippases mediate rapid, bidirectional, and rather unspecific phospholipid flip-flop with half-times of minutes or less (4 -7). A similar flippase activity was also found in the bacterial inner membrane, where lipid synthesis occurs likewise at the cytoplasmic leaflet (8).Techniques to determine transbilayer phospholipid movement as well as the activity of flippases have been critically evaluated (9). Spin-labeled and fluorescent lipid analogues have provided much insight into protein-mediated transbilayer dynamics of phospholipids (9). However, the bulky reporter moieties may affect the absolute values of transbilayer lipid movement.An alternati...

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supporting

confidence: 67%

Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

Papadopulos

Vehring

López‐Montero

et al. 2007

Journal of Biological Chemistry

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“…Midexponentially grown cells of AD or its transformants, expressing CDR1, CDR2 or CDR3, when incubated with donor vesicles containing either M-C 6 NBD-PE, M-C 6 NBD-PS or M-C 6 NBD-PC, led to transfer of rapidly exchangeable NBD lipid analogues from the quenched environment of the donor vesicles to the non-quenched environment of the cells (Figure 2, steps A, B). NBD analogues did not show any fluorescence in donor vesicles alone, due to presence of a phospholipid (N-Rh-DOPE) containing a different fluorophore, which has a higher excitation wavelength, thus quenching NBD fluorescence by a resonance energy transfer mechanism (Hrafnsdottir et al, 1997). N-Rh-DOPE, being a 'non-exchangeable phospholipid', does not participate in the donor-vesicle-to-celllipid exchange process due to its bulky fluorophore, covalently attached to the polar head moiety of the lipid molecule (Struck and Pagano, 1980).…”

Section: Labelling Of Plasma Membrane Of Ad and Its Transformants Witmentioning

confidence: 99%

ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators

Smriti

Krishnamurthy

Dixit

et al. 2002

Yeast

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We have used fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-tagged phospholipid analogues, NBD-PE (phosphatidylethanolamine), NBD-PC (phosphatidylcholine) and NBD-PS (phosphatidylserine), to demonstrate that Cdr1p and its other homologues, Cdr2p and Cdr3p, belonging to the ATP-binding cassette (ABC) superfamily behave as general phospholipid translocators. Interestingly, CDR1 and CDR2, whose overexpression leads to azole resistance in C. albicans, elicit in-to-out transbilayer phospholipid movement, while CDR3, which is not involved in drug resistance, carries out-to-in translocation of phospholipids between the two monolayers of plasma membrane. Cdr1p, Cdr2p and Cdr3p could be further distinguished on the basis of their sensitivities to different inhibitors. For example, the in-to-out activity associated with Cdr1p and Cdr2p is energy-dependent and sensitive to sulphydryl blocking agents such as N-ethylmaleimide (NEM) and cytoskeleton disrupting agent cytochalasin E, while Cdr3p-associated out-to-in activity is energydependent but insensitive to NEM and cytochalasin E. We found that certain drugs, such as fluconazole, cycloheximide and miconazole, to which Cdr1p confers resistance could also affect in-to-out transbilayer movement of NBD-PE, while the same drugs had no effect on Cdr3p-mediated out-to-in translocation of NBD-PE. The ineffectiveness of these drugs to affect Cdr3p mediated out-to-in phospholipid translocation further confirms the inherent difference in the directionality of phospholipid translocation between these pumps. Notwithstanding the role of some of the Cdrps in drug resistance, this study clearly demonstrates that these ABC transporters of C. albicans are phospholipid translocators and this function could represent one of the physiological functions of such large family of proteins.

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“…This "big beast" has been identified as an experimental organism for studies on various cell structures and functions [1,2]. Its ecological and economical value has been established and reviewed [3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers forphaCGene ofBacillus megaterium

et al. 2013

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Bacillus megaterium is gaining recognition as an experimental model and biotechnologically important microorganism. Recently, descriptions of new strains of B. megaterium and closely related species isolated from diverse habitats have increased. Therefore, its identification requires several tests in combination which is usually time consuming and difficult to do. We propose using the uniqueness of the polyhydroxyalkanoate synthase C gene of B. megaterium in designing primers that amplify the 0.9 kb region of the phaC for its identification. The PCR method was optimized to amplify 0.9 kb region of phaC gene. After optimization of the PCR reaction, two methods were investigated in detail. Method I gave an amplification of a single band of 0.9 kb only in B. megaterium and was demonstrated by several strains of B. megaterium isolated from different habitats. The use of Method I did not result in the amplification of the phaC gene with other members of Bacillales. The specificity for identification of B. megaterium was confirmed using sequencing of amplicon and RT-PCR. Method II showed multiple banding patterns of nonspecific amplicons among polyhydroxyalkanoate accumulating members of Bacillales unique to the respective species. These methods are rapid and specific for the identification of polyhydroxyalkanoate accumulating B. megaterium and members of Bacillales.

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Transbilayer Movement of Fluorescent Phospholipids in Bacillus megaterium Membrane Vesicles

Cited by 61 publications

References 31 publications

Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers forphaCGene ofBacillus megaterium

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