Phosphatidylserine translocation at the yeast<i>trans</i>-Golgi network regulates protein sorting into exocytic vesicles

Hankins, Hannah M.; Sere, Yves Y.; Diab, Nicholas S.; Menon, Anant K.; Graham, Todd R.

doi:10.1091/mbc.e15-07-0487

Cited by 62 publications

(65 citation statements)

References 86 publications

(117 reference statements)

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“…0.1 A 600 eq of cell lysate was subjected to SDS-gel electrophoresis. Western blotting analysis was performed using anti-GFP antibody (1:2000) (57) to distinguish between cleaved GFP (26 kDa) and intact GFP-tagged Rer1 (40 kDa). For quantitation, membranes were scanned at 800 nm using the Odyssey Infrared Imaging System; median GFP intensities were quantified using Odyssey software (LI-COR Biosciences).…”

Section: Neo1 Influences Plasma Membrane Asymmetrymentioning

confidence: 99%

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The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane

Takar

Graham

2016

Journal of Biological Chemistry

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Eukaryotic organisms typically express multiple type IV P-type ATPases (P4-ATPases), which establish plasma membrane asymmetry by flipping specific phospholipids from the exofacial to the cytosolic leaflet. Saccharomyces cerevisiae, for example, expresses five P4-ATPases, including Neo1, Drs2, Dnf1, Dnf2, and Dnf3. Neo1 is thought to be a phospholipid flippase, although there is currently no experimental evidence that Neo1 catalyzes this activity or helps establish membrane asymmetry. Here, we use temperature-conditional alleles (neo1 ts ) to test whether Neo1 deficiency leads to loss of plasma membrane asymmetry. Wild-type (WT) yeast normally restrict most of the phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the inner cytosolic leaflet of the plasma membrane. However, the neo1-1 ts and neo1-2 ts mutants display a loss of PS and PE asymmetry at permissive growth temperatures as measured by hypersensitivity to pore-forming toxins that target PS (papuamide A) or PE (duramycin) exposed in the extracellular leaflet. When shifted to a semi-permissive growth temperature, the neo1-1 ts mutant became extremely hypersensitive to duramycin, although the sensitivity to papuamide A was unchanged, indicating preferential exposure of PE. This loss of asymmetry occurs despite the presence of other flippases that flip PS and/or PE. Even when overexpressed, Drs2 and Dnf1 were unable to correct the loss of asymmetry caused by neo1 ts . However, modest overexpression of Neo1 weakly suppressed loss of membrane asymmetry caused by drs2⌬ with a more significant correction of PE asymmetry than PS. These results indicate that Neo1 plays an important role in establishing PS and PE plasma membrane asymmetry in budding yeast.

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Section: Neo1 Influences Plasma Membrane Asymmetrymentioning

confidence: 99%

“…Subcellular Fractionation-Cell lysates were analyzed by subcellular fractionation as described previously (57). Seven fractions in total were collected from the top of the sucrose gradient.…”

Section: Neo1 Influences Plasma Membrane Asymmetrymentioning

confidence: 99%

The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane

Takar

Graham

2016

Journal of Biological Chemistry

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“…Hankins and collaborators have demonstrated that the plasma membrane proteins Pmal and Canl were missorted to vacuoles in S. cerevisiae drs2A cells [34], indicating that lipid flippases may play a role in sorting specific cargoes into vesicles. In our study, the morphological analysis of aptl Δ cells by TEM demonstrates that lack of Aptl resulted in abnormal organization of endomembranes in C. neoformans.…”

Section: Discussionmentioning

confidence: 99%

The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans

Rizzo

Colombo

Zamith-Miranda

et al. 2018

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.

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“…Jointly, the studies on Osh4p and OSBP suggest how sterols can be enriched directly at the trans-Golgi and why these proteins contribute significantly to the profound lipid reorganization that occurs in this compartment and beyond [12,22]. Interestingly, lipidomic analysis in yeast indicates that the percentage of sterol increases from 10 to 20 % between the trans-Golgi and the Golgi-derived exocytic vesicles [23], suggesting that these vesicles are sterol conveyors and contribute to sterol build-up in the PM.…”

Section: Using a Ptdins(4)p Gradient To Create A Sterol Gradientmentioning

confidence: 99%

New molecular mechanisms of inter-organelle lipid transport

Drin

Filseck

Čopič

2016

Biochemical Society Transactions

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Lipids are precisely distributed in cell membranes, along with associated proteins defining organelle identity. Because the major cellular lipid factory is the endoplasmic reticulum (ER), a key issue is to understand how various lipids are subsequently delivered to other compartments by vesicular and non-vesicular transport pathways. Efforts are currently made to decipher how lipid transfer proteins (LTPs) work either across long distances or confined to membrane contact sites (MCSs) where two organelles are at close proximity. Recent findings reveal that proteins of the oxysterol-binding protein related-proteins (ORP)/oxysterol-binding homology (Osh) family are not all just sterol transporters/sensors: some can bind either phosphatidylinositol 4-phosphate (PtdIns(4)P) and sterol or PtdIns(4)P and phosphatidylserine (PS), exchange these lipids between membranes, and thereby use phosphoinositide metabolism to create cellular lipid gradients. Lipid exchange is likely a widespread mechanism also utilized by other LTPs to efficiently trade lipids between organelle membranes. Finally, the discovery of more proteins bearing a lipid-binding module (SMP or START-like domain) raises new questions on how lipids are conveyed in cells and how the activities of different LTPs are coordinated.

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Phosphatidylserine translocation at the yeasttrans-Golgi network regulates protein sorting into exocytic vesicles

Cited by 62 publications

References 86 publications

The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane

The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane

The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans

New molecular mechanisms of inter-organelle lipid transport

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