scholarly journals Synthesis of Sphingolipids with Very Long Chain Fatty Acids but Not Ergosterol Is Required for Routing of Newly Synthesized Plasma Membrane ATPase to the Cell Surface of Yeast

2005 ◽  
Vol 280 (23) ◽  
pp. 22515-22522 ◽  
Author(s):  
Barbara Gaigg ◽  
Birgit Timischl ◽  
Linda Corbino ◽  
Roger Schneiter
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Chain Fatty Acid ◽  
Proton Pumping ◽  
Sterol Synthesis ◽  
Plasma Membrane Atpase ◽  
Long Chain Fatty Acid ◽  
Long Chain

The proton pumping H+-ATPase, Pma1p, is an abundant and very long-lived polytopic protein of the Saccharomyces cerevisiae plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as an excellent model to study plasma membrane biogenesis. We have previously shown that newly synthesized Pma1p is mistargeted to the vacuole in an elo3Δ mutant that affects the synthesis of the ceramide-bound C26 very long chain fatty acid (Eisenkolb, M., Zenzmaier, C., Leitner, E., and Schneiter, R. (2002) Mol. Biol. Cell 13, 4414–4428) and now describe a more detailed analysis of the role of lipids in Pma1p biogenesis. Remarkably, a block at various steps of sterol biosynthesis, a complete block in sterol synthesis, or the substitution of internally synthesized ergosterol by externally supplied ergosterol or even by cholesterol does not affect Pma1p biogenesis or its association with detergent-resistant membrane domains (lipid “rafts”). However, a block in sphingolipid synthesis or any perturbation in the synthesis of the ceramide-bound C26 very long chain fatty acid results in mistargeting of newly synthesized Pma1p to the vacuole. Mistargeting correlates with a lack of newly synthesized Pma1p to acquire detergent resistance, suggesting that sphingolipids with very long acyl chains affect sorting of Pma1p to the cell surface.

scholarly journals A Specific Structural Requirement for Ergosterol in Long-chain Fatty Acid Synthesis Mutants Important for Maintaining Raft Domains in Yeast

2002 ◽  
Vol 13 (12) ◽  
pp. 4414-4428 ◽  
Author(s):  
Marlis Eisenkolb ◽  
Christoph Zenzmaier ◽  
Erich Leitner ◽  
Roger Schneiter
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Double Mutant ◽  
Chain Fatty Acid ◽  
Long Chain Fatty Acid ◽  
Structural Requirement ◽  
Fatty Acid Elongase ◽  
Synthetically Lethal ◽  
Raft Domains ◽  
Long Chain

Fungal sphingolipids contain ceramide with a very-long-chain fatty acid (C26). To investigate the physiological significance of the C26-substitution on this lipid, we performed a screen for mutants that are synthetically lethal with ELO3. Elo3p is a component of the ER-associated fatty acid elongase and is required for the final elongation cycle to produce C26 from C22/C24 fatty acids.elo3Δ mutant cells thus contain C22/C24- instead of the natural C26-substituted ceramide. We now report that under these conditions, an otherwise nonessential, but also fungal-specific, structural modification of the major sterol of yeast, ergosterol, becomes essential, because mutations in ELO3 are synthetically lethal with mutations in ERG6. Erg6p catalyzes the methylation of carbon atom 24 in the aliphatic side chain of sterol. The lethality of an elo3Δ erg6Δ double mutant is rescued by supplementation with ergosterol but not with cholesterol, indicating a vital structural requirement for the ergosterol-specific methyl group. To characterize this structural requirement in more detail, we generated a strain that is temperature sensitive for the function of Erg6p in an elo3Δ mutant background. Examination of raft association of the GPI-anchored Gas1p and plasma membrane ATPase, Pma1p, in the conditional elo3Δ erg6 ts double mutant, revealed a specific defect of the mutant to maintain raft association of preexisting Pma1p. Interestingly, in an elo3Δ mutant at 37°C, newly synthesized Pma1p failed to enter raft domains early in the biosynthetic pathway, and upon arrival at the plasma membrane was rerouted to the vacuole for degradation. These observations indicate that the C26 fatty acid substitution on lipids is important for establishing raft association of Pma1p and stabilizing the protein at the cell surface. Analysis of raft lipids in the conditional mutant strain revealed a selective enrichment of ergosterol in detergent-resistant membrane domains, indicating that specific structural determinants on both sterols and sphingolipids are required for their association into raft domains.

scholarly journals The effect of intracellular pH on long-chain fatty acid uptake in 3T3-L1 adipocytes: evidence that uptake involves the passive diffusion of protonated long-chain fatty acids across the plasma membrane

1996 ◽  
Vol 313 (2) ◽  
pp. 487-494 ◽  
Author(s):  
Bernardo L. TRIGATTI ◽  
Gerhard E. GERBER
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Chain Fatty Acid ◽  
Long Chain Fatty Acids ◽  
Cytoplasmic Ph ◽  
Long Chain Fatty Acid ◽  
Intact Cells ◽  
Fatty Acid Binding ◽  
Long Chain

To understand the mechanism of long-chain fatty acid permeation of the plasma membrane in mammalian cells, the effects of changes in the cytoplasmic pH on the internalization of physiologically relevant, submicromolar concentrations of uncomplexed long-chain fatty acids were investigated in 3T3-L1 adipocytes. The acidification of the cytoplasm upon NH4Cl prepulsing of intact cells was accompanied by a rapid reduction of cellular long-chain fatty acid uptake (measured as the total accumulation of [9,10-3H]oleate). This was followed by a slow recovery to normal levels of uptake as the cytoplasmic pH recovered. Conventional filtration assays do not distinguish between fatty acid movement across the plasma membrane and intracellular steps, such as binding to cytoplasmic fatty acid-binding proteins or metabolism. While the in vitro binding of a photoreactive fatty acid, 11-m-diazirinophenoxy[11-3H]undecanoate, to a cytoplasmic fatty acid-binding protein was insensitive to changes in pH from pH 7.5 to 5.5, the in vitro conversion of oleate into oleoyl-CoA by cellular acyl-CoA synthetase decreased dramatically. Therefore, the labelling of the 15 kDa cytoplasmic fatty acid-binding protein in intact cells by the photoreactive fatty acid was used as a more direct measure of the permeation of the probe across the plasma membrane. Acidification of the cytoplasm resulted in an immediate reduction in the labelling of this protein in intact adipocytes. Its photolabelling recovered, however, upon the recovery of the cytoplasmic pH to normal levels. This was due to effects of the cytoplasmic pH on the permeation of the photoreactive fatty acid across the plasma membrane rather than its binding to the 15 kDa protein or metabolism in vivo. This is the first demonstration that the movement of physiologically relevant, submicromolar concentrations of uncomplexed long-chain fatty acids across the plasma membrane of intact cells is coupled to the cytoplasmic pH and suggests that it occurs by the diffusion of the protonated long-chain fatty acid through the lipid bilayer.

scholarly journals FAT/CD36-mediated Long-Chain Fatty Acid Uptake in Adipocytes Requires Plasma Membrane Rafts

2005 ◽  
Vol 16 (1) ◽  
pp. 24-31 ◽  
Author(s):  
Jürgen Pohl ◽  
Axel Ring ◽  
Ümine Korkmaz ◽  
Robert Ehehalt ◽  
Wolfgang Stremmel
Keyword(s):  
Plasma Membrane ◽  
Oleic Acid ◽  
Fatty Acid ◽  
Lipid Rafts ◽  
Chain Fatty Acid ◽  
Dominant Negative Mutant ◽  
Acid Uptake ◽  
Long Chain Fatty Acid ◽  
Lcfa Uptake ◽  
Long Chain

We previously reported that lipid rafts are involved in long-chain fatty acid (LCFA) uptake in 3T3-L1 adipocytes. The present data show that LCFA uptake does not depend on caveolae endocytosis because expression of a dominant negative mutant of dynamin had no effect on uptake of [3H]oleic acid, whereas it effectively prevented endocytosis of cholera toxin. Isolation of detergent-resistant membranes (DRMs) from 3T3-L1 cell homogenates revealed that FAT/CD36 was expressed in both DRMs and detergent-soluble membranes (DSMs), whereas FATP1 and FATP4 were present only in DSMs but not DRMs. Disruption of lipid rafts by cyclodextrin and specific inhibition of FAT/CD36 by sulfo-N-succinimidyl oleate (SSO) significantly decreased uptake of [3H]oleic acid, but simultaneous treatment had no additional or synergistic effects, suggesting that both treatments target the same mechanism. Indeed, subcellular fractionation demonstrated that plasma membrane fatty acid translocase (FAT/CD36) is exclusively located in lipid rafts, whereas intracellular FAT/CD36 cofractionated with DSMs. Binding assays confirmed that [3H]SSO predominantly binds to FAT/CD36 within plasma membrane DRMs. In conclusion, our data strongly suggest that FAT/CD36 mediates raft-dependent LCFA uptake. Plasma membrane lipid rafts might control LCFA uptake by regulating surface availability of FAT/CD36.

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