The lipids of enveloped viruses play critical roles in viral morphogenesis and infectivity. They are derived from the host membranes from which virus budding occurs, but the precise lipid composition has not been determined for any virus. Employing mass spectrometry, this study provides a quantitative analysis of the lipid constituents of HIV and a comprehensive comparison with its host membranes. Both a substantial enrichment of the unusual sphingolipid dihydrosphingomyelin and a loss of viral infectivity upon inhibition of sphingolipid biosynthesis in host cells are reported, establishing a critical role for this lipid class in the HIV replication cycle. Intriguingly, the overall lipid composition of native HIV membranes resembles detergent-resistant membrane microdomains and is strikingly different from that of host cell membranes. With this composition, the HIV lipidome provides strong evidence for the existence of lipid rafts in living cells.dihydrosphingomyelin ͉ nano-electrospray ionization tandem mass spectrometry ͉ lipid analysis ͉ viral membrane

Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or "rafts" on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (>3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ϳ20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure.The separation of membrane lipids into different phases creates diverse microenvironments within a biological membrane (1, 2). In particular, cholesterol is believed to condense with saturated phosphatidylcholine (PC) 1 and sphingomyelin (SM) to form minute patches (40 -100 nm wide) of lipids in a liquid-ordered phase (3-7), creating specialized lipid microenvironments called "rafts" within the disordered fluid phase formed by unsaturated lipids (8). These ordered microdomains control the access and egress of subsets of membrane proteins, regulating signaling systems at the cell surface (9). liquidordered domains resist solubilization in non-ionic detergents (6, 7, 10 -13), enabling them to be isolated as detergent-resistant membranes (DRMs) that float at low density upon gradient centrifugation (14). Lipid-anchored proteins partition into both leaflets of these domains, the glycosylphosphatidylinositol (GPI)-anchored proteins into the outer (surface) layer and the diacylated cytoplasmic proteins into the inner layer (9, 15, 16). The membrane environment of GPI-anchored prion protein (PrP) is of particular interest since it is a candidate for chaperoning the conversion of PrP to the altered pathogenic conformation associated with prion disease (17-19). Immunolabeling shows PrP to be present on the neuronal surface in different, albeit often closely adjacent, d...

A 4.8 kb DNA‐fragment was cloned and sequenced encompassing the structural gene of PFL‐deactivase (2.7 kb) and 2 kb of the 5 flanking region that contains the elements for anaerobic induction. A mutant lacking deactivase was shown to require exogenous electron acceptors for anaecrobic growth with glucose. This revealed the identity of PFL‐deactivase with the alcohol and acetaldehyde dehydrogenases of E. coli. The multienzyme represents a homopolymeric protein (∼ 40 × 96 kDa) requiring Fe2+ for all functions.

Background: The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition.

Iron-sulfur proteins acquire their clusters by posttranslational assembly. To identify components involved in this process an in vitro assay for holoprotein formation was established using the [2Fe-2S] ferredoxin of the cyanobacterium Synechocystis as a model. Conversion of apoferredoxin to the holo-form was observed in an anaerobic reaction medium containing Fe(NH 4 ) 2 (SO 4 ) 2 , L-cysteine, glutathione, and catalytic amounts of Synechocystis extract, specifically depleted of endogeneous ferredoxin. An approximate 2500-fold purification of the converter activity yielded a monomeric, 43-kDa, pyridoxal phosphate-containing enzyme, which catalyzed the breakdown of L-cysteine to yield sulfide (assembled in ferredoxin), pyruvate, and ammonia; 1 mol of [2Fe-2S] ferredoxin was formed per 2 mol of cysteine utilized.The purified enzyme also catalyzed the ␤-elimination reaction with cysteine in the absence of apoferredoxin. An increased reactivity was found with cystine instead of cysteine, which should yield cysteine persulfide as the primary product.These results provide a function-based identification of a cysteine/cystine C-S-lyase as a participant in ferredoxin Fe-S cluster formation. A substrate-derived cysteine persulfide could be involved in this reaction.Iron-sulfur (Fe-S) clusters are found as functional units in numerous electron-transferring proteins. They are essential in basic biological processes such as oxidative phosphorylation, photosynthesis, and nitrogen fixation. More recently, they have also been identified as components of enzymes not concerned with redox reactions (1). Lately they have been implicated in gene regulation (2).Despite these important and diverse functions and thorough biochemical investigations of Fe-S proteins, the biosynthetic steps leading to Fe-S cluster assembly have not been established. While it seems likely that enzyme-catalyzed reactions are involved in the formation of Fe-S clusters in vivo, the chemical reconstitution of apoproteins with free ferrous ion and sulfide in the presence of thiols (3) has been most intensively exploited for cluster build-up in vitro. Nevertheless, several proteins have been suggested as candidates in the biosynthetic process. Rhodanese (4) and 3-mercaptopyruvate sulfurtransferase (5) were effective in purified in vitro systems, comprising apoprotein, thiol, ferrous ion, and the respective sulfur-containing substrate, thiosulfate, or mercaptopyruvate. Analysis of yeast respiration-deficient mutants suggested a role for the BCS1 gene product (6) in the synthesis of functional Rieske protein. Studies on nif mutants of Azotobacter vinelandii (7) identified nifS as essential for nitrogenase activity. More recently, nifS was cloned and expressed in Escherichia coli; the gene product (NifS) was characterized as a pyridoxal phosphate-containing L-cysteine desulfurase, which yielded alanine and elemental sulfur as products (8); with dithiothreitol present in the reaction mixture, sulfide was produced instead of sulfur. Further work showed that the apo-...