J. Wylie Nichols

Comparison of mechanisms of anemia in mice with sickle cell disease and β-thalassemia: Peripheral destruction, ineffective erythropoiesis, and phospholipid scramblase-mediated phosphatidylserine exposure

OBJECTIVES 1). To study the mechanisms of anemia, erythroid hyperplasia, and red blood cell (RBC) clearance in murine models of sickle cell disease (Sickle) and beta-thalassemia (Th1/Th1); 2) To determine the contribution of the phospholipid scramblase enzyme to phosphatidylserine (PS) exposure and RBC death in Sickle and Th1/Th1 mice. METHODS We used a combination of flow-cytometric analysis and assays for phospholipid remodeling to determine the extent and sites of erythroid hyperplasia, PS exposure, and cell death. RESULTS 1) Sickle RBCs have a much shorter half-life than Th1/Th1 RBCs (0.8 days vs. 11 days). A significant proportion of Th1/Th1 peripheral reticulocytes mature into erythrocytes, however, approximately fivefold fewer Sickle reticulocytes mature. While erythroid hyperplasia exists in both Sickle and Th1/Th1 mice, Th1/Th1 produce fourfold more RBCs than necessary to maintain steady state, while Sickle produce no excess RBCs. 2) 61% of Sickle and 34% of Th1/Th1 RBCs are scramblase(+) as measured by internalization assays of the fluorescent phospholipid NBD-PC. The majority of NBD-PC(+) RBCs are also annexin-V(+), supporting a mechanistic link between scramblase activity and PS exposure. A proportion of both reticulocytes and older RBCs in Sickle and Th1/Th1 mice have active scramblase, and the degree of scramblase activation in these strains correlates with the propensity for RBC death. CONCLUSIONS Sickle and Th1/Th1 mice are both anemic, with significant erythroid hyperplasia. Th1/Th1 mice display ineffective erythropoiesis while Sickle mice show rapid peripheral destruction of RBCs. PS exposure and phospholipid scramblase activity serve as markers of RBCs with altered phospholipid asymmetry and greater propensity for cell death.

NBD-Labeled Phosphatidylcholine and Phosphatidylethanolamine are Internalized by Transbilayer Transport across the Yeast Plasma Membrane

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M‐C6‐NBD‐PC) and phosphatidylethanolamine (M‐C6‐NBD‐PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M‐C6‐NBD‐PC was internalized predominantly to the vacuole and degraded. M‐C6‐NBD‐PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2°C, both were internalized to the nuclear envelope/ER and mitochondria by an energy‐dependent, N‐ethylmaleimide‐sensitive process, and transport of M‐C6‐NBD‐PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis‐defective mutant, end4‐1. However, following pre‐incubation at 37°C, internalization of both phospholipids was inhibited at 2°C and 37°C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M‐C6‐NBD‐PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M‐C6‐NBD‐PC and M‐C6‐NBD‐PE are transported across the plasma membrane to the cytosolic leaflet by a protein‐mediated, energy‐dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M‐C6‐NBD‐PC, but not M‐C6‐NBD‐PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.

Saccharomyces cerevisiae Npc2p Is a Functionally Conserved Homologue of the Human Niemann-Pick Disease Type C 2 Protein, hNPC2

ABSTRACT Niemann-Pick Disease Type C (NP-C) is a fatal neurodegenerative disease, which is biochemically distinguished by the lysosomal accumulation of exogenously derived cholesterol. Mutation of either the hNPC1 or hNPC2 gene is causative for NP-C. We report the identification of the yeast homologue of human NPC2, Saccharomyces cerevisiae Npc2p. We demonstrate that scNpc2p is evolutionarily related to the mammalian NPC2 family of proteins. We also show, through colocalization, subcellular fractionation, and secretion analyses, that yeast Npc2p is treated similarly to human NPC2 when expressed in mammalian cells. Importantly, we show that yeast Npc2p can efficiently revert the unesterified cholesterol and GM1 accumulation seen in hNPC2−/− patient fibroblasts demonstrating that it is a functional homologue of human NPC2. The present study reveals that the fundamental process of NPC2-mediated lipid transport has been maintained throughout evolution.

A yeast model system for functional analysis of the Niemann-Pick type C protein 1 homolog, Ncr1p.

Niemann–Pick disease type C (NP‐C) is a progressive, ultimately fatal, autosomal recessive neurodegenerative disorder. The major biochemical hallmark of the disease is the endocytic accumulation of low‐density lipoprotein‐derived cholesterol. The majority of NP‐C patients have mutations in the Niemann‐Pick type C1 gene, NPC1. This study focuses on the Saccharomyces cerevisiae homolog of the human NPC1 protein encoded by the NCR1 gene. Ncr1p localizes to the vacuole, the yeast equivalent to the mammalian endosome‐lysosome system. Here, we identify the first phenotype caused by deletion of NCR1 from the yeast genome, resistance to the ether lipid drug, edelfosine. Our results indicate that edelfosine has a cytotoxic, rather than cytostatic, effect on wildtype yeast cells. We exploit the edelfosine resistance phenotype to assess the function of yeast Ncr1 proteins carrying amino acid changes corresponding to human NPC1 patient mutations. We find that one of these amino acid changes severely compromises Ncr1p function as assessed using the edelfosine resistance assay. These findings establish S. cerevisiae as a model system that can be exploited to analyze the molecular consequences of patient mutations in NPC1 and provide the basis for future genetic studies using yeast.

Fluorescent, Acyl Chain-labeled Phosphatidylcholine Analogs Reveal Novel Transport Pathways across the Plasma Membrane of Yeast

Acyl chain-labeled NBD-phosphatidylcholine (NBD-PC) has been used to identify three gene products (Lem3p, Dnf1p, and Dnf2p) that are required for normal levels of inward-directed phospholipid transport (flip) across the plasma membrane of yeast. Although the head group structure of acyl chain-labeled NBD phospholipids has been shown to influence the mechanism of flip across the plasma membrane, the extent to which the acyl chain region and the associated fluorophore affect flip has not been assessed. Given the identification of these proteins required for NBD-PC flip, it is now possible to determine whether the fluorophore attached to a phospholipid acyl chain influences the mechanism of flip. Thus, flip of phosphatidylcholine molecules with three different Bodipy fluorophores (Bodipy FL, Bodipy 530, and Bodipy 581) was tested and compared with that of NBD-PC in strains carrying deletions in LEM3, DNF1, and DNF2. Deletion of these genes significantly reduced the flip of NBD-PC and Bodipy FL-PC but had no effect on that of Bodipy 581-PC and Bodipy 530-PC. These data, in combination with comparisons of the effect of ATP depletion, collapse of the proton electrochemical gradient across the plasma membrane, and culture density led to the conclusion that at least three different flip pathways exist in yeast that are selective for the structure of the fluorophore attached to the acyl chain of phosphatidylcholine molecules.

The putative aminophospholipid translocases, DNF1 and DNF2, are not required for 7-nitrobenz-2-oxa-1,3-diazol-4-yl-phosphatidylserine flip across the plasma membrane of Saccharomyces cerevisiae.

The regulation of phosphatidylserine (PS) distribution across the plasma membrane of eukaryotic cells has been implicated in numerous cell functions (e.g. apoptosis and coagulation). In a recent study, fluorescent phospholipids labeled in the acyl chain with 7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD) were used to identify two members of the P4 subfamily of P-type ATPases, Dnf1p and Dnf2p, that are necessary for the inward-directed transport of phospholipids across the plasma membrane (flip) of yeast ( Pomorski, T., Lombardi, R., Riezman, H., Devaux, P. F., Van Meer, G., and Holthuis, J. C. (2003) Mol. Biol. Cell 14, 1240-1254 ). Herein, we present evidence that the flip of NBD-labeled PS (NBD-PS) across the plasma membrane does not require the expression of Dnf1p or Dnf2p. In strains in which DNF1 and DNF2 are both deleted, the flip of NBD-PS is increased ∼2-fold over that of the isogenic parent strain, whereas the flip of NBD-labeled phosphatidylcholine and NBD-labeled phosphatidylethanolamine are reduced to ∼20 and ∼50%, respectively. The mechanism responsible for NBD-PS flip is similar to that for NBD-labeled phosphatidylcholine and NBD-labeled phosphatidylethanolamine in its dependence on cellular ATP and the plasma membrane proton electrochemical gradient, as well as its regulation by the transcription factors Pdr1p and Pdr3p. Based on the observation that deletion or inactivation of all four members of the DRS2/DNF essential subfamily of P-type ATPases does not affect NBD-PS flip, we conclude that the activity reflected by NBD-PS internalization is not the essential function of the DRS2/DNF subfamily of P-type ATPases.

The Proton Electrochemical Gradient across the Plasma Membrane of Yeast Is Necessary for Phospholipid Flip

Recently, two members of the P4 family of P-type ATPases, Dnf1p and Dnf2p, were shown to be necessary for the internalization (flip) of fluorescent, 7-nitrobenz-2-oxa-1,3-diazol-4-yl(NBD)-labeled phospholipids across the plasma membrane of Saccharomyces cerevisiae. In the current study, we have demonstrated that ATP hydrolysis is not sufficient for phospholipid flip in the absence of the proton electrochemical gradient across the plasma membrane. This requirement was demonstrated by two independent means. First, collapse of the plasma membrane proton electrochemical gradient by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) almost completely blocked NBD-phospholipid flip while only moderately reducing the cytosolic ATP concentration. Second, strains with point mutations in PMA1, which encodes the plasma membrane proton pump that generates the proton electrochemical gradient, are defective in NBD-PC flip, whereas their cytosolic ATP content is actually increased. These results establish that the proton electrochemical gradient is required for NBD-phospholipid flip across the plasma membrane of yeast and raise the question whether it contributes an additional required driving force or whether it functions as a regulatory signal.

Internalization and trafficking of fluorescent-labeled phospholipids in yeast

Phospholipid reporter molecules, containing a fluorescent group attached to a short, acyl chain, spontaneously insert into the plasma membrane of yeast cells allowing retrograde trafficking to intracellular organelles as well as their metabolic fates to be monitored. This approach provides the framework for determining the dependence of particular phospholipid trafficking and metabolic steps on a wide range of genes known to be required for related membrane transport functions as well as for developing genetic screens to identify novel genes required for these processes. This review presents an overview of insights gained into phospholipid trafficking and metabolism using this approach.

Mechanisms of Proton-Hydroxide Flux Across Membranes

In both model and biological membranes, measurements of proton-hydroxide flux produce permeability coefficients that are orders of magnitude greater than those obtained with other monovalent ions. As a working hypothesis, we suggest that this large variance can be explained by the flux of protons along hydrogenbonded strands of associated water or protein in the hydrophobic plane of the membrane.