A Novel Pathway for Transport and Metabolism of a Fluorescent Phosphatidic Acid Analog in Yeast

Abstract

The various membranes of a cell have distinctive lipid compositions and membrane structures that are necessary for the specific functions of each membrane and compartmentalization of biochemical pathways. Movement of phospholipids between and within membranes is essential for the development and maintenance of this specialized membrane organization. A vast amount of data have been generated toward describing processes of intracellular lipid transport, revealing that lipids of the various classes are transported via diverse mechanisms (1 -3). Despite this significant body of knowledge, however, identification of the molecular mediators of these processes has, for the most part, remained elusive.Many methods have been employed to monitor intracellular lipid transport (1 -3). Pagano and colleagues (4 -6) pioneered using phospholipids fluorescently labeled at the sn-2 position with N-4-nitrobenz-2-oxa-1, 3-diazole-aminocaproic acid (C 6 -NBD) to monitor the intracellular transport of several phospholipids. These lipid analogs have some aqueous solubility, so they readily partition from vesicles to the plasma membrane of cells. Internalization, or retrograde transport, and metabolism of the fluorophore can then be monitored by fluorescence microscopy and spectroscopy and their metabolism can be analyzed by routine biochemical methods. The cellular trafficking of NBD-lipids is generally considered to provide an accurate reflection of the movement of authentic lipids of the same class within the cell (3,7 -11).The yeast Saccharomyces cerevisiae has become an increasingly important model system for investigating basic cellular processes, including the cell cycle (12), secretion (13) and intracellular phospholipid transport (14 -18). Utilization of NBD-labeled lipids to study lipid transport has recently been extended to yeast by Nichols and colleagues. NBDphosphatidylcholine [NBD-PtdCho; (10)] and NBD-phosphatidylethanolamine [NBD-PtdEtn; (11)] were introduced to yeast and retrograde transport was monitored by video-enhanced microscopy. These researchers went on to devise a photo-killing technique to isolate mutants unable to internalize the NBD-PtdEtn and identified two transcription factors that regulate the transport of NBD-PtdEtn across the plasma membrane (11).Phosphatidic acid (PtdOH) and diacylglycerol (DAG) are important bioactive lipids that can be produced in the plasma membrane during cellular signaling (19,20) and have been implicated as regulators of membrane traffic (21). Recognizing the importance of understanding how these lipids are transported, Pagano and colleagues (5,7,22) examined the uptake and retrograde transport of NBD-labeled phosphatidic acid (NBD-PtdOH) in fibroblasts. Their results indicated that movement of the NBD-PtdOH from the outer leaflet of the plasma membrane across the bilayer required its dephosphorylation to NBD-DAG. Then, following passive transbilayer movement to the inner leaflet of the plasma membrane, a facilitated transport mechanism transferred the NBD-DAG to intracellular ...

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

“…At this temperature, Ͼ80% of the internalized lipid could be recovered in the intact form (unpublished data). NBD-PA uptake in yeast has previously been reported to occur via a nonendocytic pathway and independent of ATP (Trotter, 2000). However, we observed that ATP-depletion of wild-type cells caused a major reduction in the uptake of both NBD-PA (82%) and NBD-PG (85%; Figure 5A and unpublished data).…”

Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARγ agonist

“…We accomplished these goals in two ways; the first was through the use of S. cerevisiae, an organism that lacks all nuclear hormone receptors and contains only a single G protein-linked receptor for mating factor. Yeast will accumulate intact anionic phospholipids from their media (30) and can be stably transfected with a PPAR␥ reporting system (18). We found (Fig.…”

Identification of a family of animal sphingomyelin synthases

“…[Methyl‐ 14 C]‐choline was from ICN Biomedicals (Irvine, CA) and L ‐3‐phosphatidyl [ N‐ methyl‐ 3 H]‐choline,1,2‐dipalmitoyl from Amersham Pharmacia (Piscataway, NJ). NBD‐DAG was produced from NBD‐PC by treatment with phospholipase C from Bacillus cereus as described (Trotter, 2000). All other lipids and chemicals were from Sigma Aldrich (St Louis, MO), as listed in Supplementary data.…”