Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

Hachiro, Takeru; Yamamoto, Takaharu; Nakano, Kenji; Tanaka, Kazuma

doi:10.1074/jbc.m112.416263

Cited by 33 publications

(37 citation statements)

References 78 publications

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“…We previously suggested that Lem3p-Dnf1p/Dnf2p are involved in the sorting of high affinity tryptophan permease Tat2p at the TGN; in the lem3 Δ mutant, Tat2p was not properly transported to the plasma membrane and missorted to the vacuole (Hachiro et al 2013). We examined the effect of the cfs1 Δ mutation on the requirement of tryptophan for growth in the lem3 Δ mutant.…”

Section: Resultsmentioning

confidence: 99%

“…We examined the effect of the cfs1 Δ mutation on the requirement of tryptophan for growth in the lem3 Δ mutant. The lem3 Δ trp1 Δ mutant shows a severe growth defect in YPDA medium containing standard concentration of tryptophan (∼100 μg/ml), and requires high concentration of tryptophan (∼300 μg/ml) for growth (Hachiro et al 2013) (Figure 5A). The cfs1 Δ mutation partially suppressed the growth defect of the lem3 Δ trp1 Δ mutant in YPDA, suggesting that the cfs1 Δ mutation suppresses Tat2p missorting caused by dysfunction of Lem3p-Dnf1p/Dnf2p at the TGN.…”

Section: Resultsmentioning

confidence: 99%

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Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast

Yamamoto

Fujimura-Kamada

Shioji

et al. 2016

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Type 4 P-type ATPases (P4-ATPases) function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), associated with the Cdc50p family noncatalytic subunit, and one monomeric flippase, Neo1p. They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified. Here, to search for novel factors that functionally interact with flippases, we screened transposon insertional mutants for strains that suppressed the cold-sensitive growth defect in the cdc50Δ mutant. We identified a mutation of YMR010W encoding a novel conserved membrane protein that belongs to the PQ-loop family including the cystine transporter cystinosin and the SWEET sugar transporters. We named this gene CFS1 (cdc fifty suppressor 1). GFP-tagged Cfs1p was partially colocalized with Drs2p and Neo1p to endosomal/late Golgi membranes. Interestingly, the cfs1Δ mutation suppressed growth defects in all flippase mutants. Accordingly, defects in membrane trafficking in the flippase mutants were also suppressed. These results suggest that Cfs1p and flippases function antagonistically in membrane trafficking pathways. A growth assay to assess sensitivity to duramycin, a phosphatidylethanolamine (PE)-binding peptide, suggested that the cfs1Δ mutation changed PE asymmetry in the plasma membrane. Cfs1p may thus be a novel regulator of phospholipid asymmetry.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast

Yamamoto

Fujimura-Kamada

Shioji

et al. 2016

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“…However, changes in membrane lipid composition and physical properties mediated by increased PI(4,5)P 2 levels may be on the basis of the endocytosis defect exhibited by the inp51 mutant. Recent results reported by Hachiro et al [33] indicate that the destination of Tat2 is regulated by the interaction of its NH 2 -terminal region with phosphatidylserine (PS) in the cytoplasmic leaflet of membranes.…”

Section: Accepted Manuscriptmentioning

confidence: 98%

Characterization of the S. cerevisiae inp51 mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content, membrane fluidity and cold growth

Córcoles-Sáez

Hernández

Martínez-Rivas

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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“…Lem3 is required for normal development of flippase activity at the plasma membrane in association with the catalytic subunits Dnf1 and Dnf2 (53). Lem3-dependent flippase activity is required for wildtype PHS resistance (11), likely through its role in the normal delivery of Tat2 to the plasma membrane (68). Mutants lacking Lem3 are highly sensitive to AbA but also extremely resistant to Myr (54,69).…”

Section: Discussionmentioning

confidence: 99%

“…7. If Tat2 is used as a representative membrane transporter, then Pdr5 and Yor1 are positive regulators of its internalization, and the Lem3-dependent flippase is a negative regulator as shown (68). A membrane transporter that could efflux AbA would be an ideal candidate for such a target of Pdr5/Yor1-and Lem3-dependent regulation.…”

Section: Discussionmentioning

confidence: 99%

Control of Plasma Membrane Permeability by ABC Transporters

et al. 2015

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h ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5⌬ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3⌬ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5⌬ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5⌬ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2.

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Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

Cited by 33 publications

References 78 publications

Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast

Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast

Characterization of the S. cerevisiae inp51 mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content, membrane fluidity and cold growth

Control of Plasma Membrane Permeability by ABC Transporters

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