Ceramide is a membrane lipid involved in a number of crucial biological processes. Recent evidence suggests that ceramide is likely to reside and function within lipid rafts; ordered sphingolipid and cholesterol-rich lipid domains believed to exist within many eukaryotic cell membranes. Using lipid vesicles containing co-existing raft domains and disordered fluid domains, we find that natural and saturated synthetic ceramides displace sterols from rafts. Other raft lipids remain raft-associated in the presence of ceramide, showing displacement is relatively specific for sterols. Like cholesterol-containing rafts, ceramide-rich "rafts" remain in a highly ordered state. Comparison of the sterol-displacing abilities of natural ceramides with those of saturated diglycerides and an unsaturated ceramide demonstrates that tight lipid packing is critical for sterol displacement by ceramide. Based on these results, and the fact that cholesterol and ceramides both have small polar headgroups, we propose that ceramides and cholesterol compete for association with rafts because of a limited capacity of raft lipids with large headgroups to accommodate small headgroup lipids in a manner that prevents unfavorable contact between the hydrocarbon groups of the small headgroup lipids and the surrounding aqueous environment. Minimizing the exposure of cholesterol and ceramide to water may be a strong driving force for the association of other molecules with rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft structure and function, altering liquid ordered properties as well as molecular composition. In this regard, certain previously observed physiological processes may be a result of displacement. In particular, a direct connection to the previously observed sphingomyelinase-induced displacement of cholesterol from plasma membranes in cells is proposed.

PAGE 4293:Fig . 3A (upper right panel) shows an alteration of a lane (10 M PD98059) that was not detected at the time of manuscript submission and that is contrary to the Journal of Biological Chemistry guidelines. Herein, we provide an alternative figure that shows that the ERK inhibitor PD98059 inhibited Vpr-(52-96)-induced ERK activation in a dose-dependent manner. THP-1 cells were treated with the indicated concentrations of PD98059 for 4 h, followed by stimulation with 1.5 M Vpr-(52-96) peptide for another 2 h. The legend of this figure remains unchanged. The data support the published observations and therefore do not impact the interpretation of this figure or the central conclusion of this article. During the continuation of our work on hsc70 trafficking, we have noticed that there was an error during the preparation of midiprep plasmid DNA for two constructs that were described in this work. The error reported by us does not alter the validity of the raw data. The following two constructs were affected by the error: GFP-hsc70(225-262) and GFP-hsc70(245-287). These two plasmid DNAs have been inverted in our article. Following the detection of the error, we have verified the correctness of all other constructs encoding wild-type or mutant fragments of domain IIB of hsc70. This error affects the presentation of results shown in Figs. 2B and 3 (A and C) and the interpretation depicted in Fig. 6A. Thus, the correct interpretation of our data is that segment 245-287 locates constitutively in the nucleolus under nonstress and stress conditions. Fragment 225-262 displays weak stressinduced nucleolar accumulation. This makes residues 225-244 the negative regulator of hsc70 nucleolar accumulation. The experiments reported were carried out at 390 mM methyl-␤-cyclodextrin (M␤CD; 825 mg ϩ 1 ml of water, which gives a volume of ϳ1.6 ml), not as reported at 625 mM M␤CD (825 mg/ml of solution). We (MiJin Son and E. L.) have investigated asymmetric vesicle preparation at the higher M␤CD concentration and found that 625 mM M␤CD could be used to produce asymmetric small unilamellar vesicles. However, the results were not as reproducible as with the lower M␤CD concentration. We recommend the use of 390 mM M␤CD to prepare asymmetric small unilamellar vesicles. The first sentence in the Abstract should read as follows: The cyclic dinucleotide c-di-AMP synthesized by the diadenylate cyclase domain was discovered recently as a messenger molecule for signaling DNA breaks in Bacillus subtilis.In the Introduction, line 21 in the right-hand column should read as follows: This group of proteins (COG3887), as represented by the B. subtilis protein YybT, contains two N-terminal transmembrane helices, a region that shares minimum sequence homology with some PerArnt-Sim (PAS) domains, a highly modified GGDEF domain, and a DHH/DHHA1 domain (see Fig. 1). ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at ...

The formation and stability of ordered lipid domains (rafts) in model membrane vesicles were studied using a series of sterols and steroids structurally similar to cholesterol. In one assay, insolubility in Triton X-100 was assessed in bilayers composed of sterol/steroid mixed with dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, or a 1:1 mixture of these phospholipids. In a second assay fluorescence quenching was used to determine the degree of ordered domain formation in bilayers containing sterol/steroid and a 1:1 mixture of DPPC and a quencher-carrying phosphatidylcholine. Both methods showed that several single modifications of the cholesterol structure weaken, but do not fully abolish, the ability of sterols and steroids to promote ordered domain formation when mixed with DPPC. Some of these modifications included a shift of the double bond from the 5-6 carbons (cholesterol) to 4-5 carbons (allocholesterol), derivatization of the 3-OH (cholesterol methyl ether, cholesteryl formate), and alteration of the 3-hydroxy to a keto group (cholestanone). An oxysterol involved in atherosclerosis, 7-ketocholesterol, formed domains with DPPC that were as thermally stable as those with cholesterol although not as tightly packed as judged by fluorescence anisotropy. It was also found that 7-ketocholesterol has fluorescence quenching properties making it a useful spectroscopic probe. Lathosterol, which has a 7-8 carbon double bond in place of the 5-6 double bond of cholesterol, formed rafts with DPPC that were at least as detergent-resistant as, and even more thermally stable than, rafts containing cholesterol. Because lathosterol is an intermediate in cholesterol biosynthesis, we conclude it is unlikely that sterol biosynthesis continues past lathosterol in order to create a raft-favoring lipid.