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Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can post-translationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533-2542). In the present study, we investigated the limits of this unassisted translocation and report that surprisingly long (85 residues) domains of different sequence and charge placed downstream of b5's TMD can posttranslationally translocate into mammalian microsomes and liposomes at nanomolar nucleotide concentrations. Furthermore, integration of these constructs occurred in vivo in translocon-defective yeast strains. Unassisted translocation was not unique to b5 but was also observed for another TA protein (protein tyrosine phosphatase 1B) whose TMD, like the one of b5, is only moderately hydrophobic. In contrast, more hydrophobic TMDs, like synaptobrevin's, were incapable of supporting unassisted integration, possibly because of their tendency to aggregate in aqueous solution. Our data resolve long-standing discrepancies on TA protein insertion and are relevant to membrane evolution, biogenesis, and physiology.

C-tail-anchored (TA) proteins often require a transmembrane domain of moderate hydrophobicity to maintain their endoplasmic reticulum residence, but the suggested role of protein--lipid interactions in this phenomenon has not been established. Here, we studied the interaction of TA proteins with lipids by differential scanning calorimetry by using a model system consisting of liposomes embedding either of two forms of cytochrome [b.sub.5]: the endoplasmic reticulum-resident wild-type ([b.sub.5]wt) and a mutant thereof ([b.sub.5]ext), that contains five extra nonpolar amino acids in its transmembrane domain and, therefore, reaches the plasma membrane. The proteins were incorporated into liposomes of palmitoyloleyl-phosphatidylcholine (POPC) or POPC mixed with either distearoyl-phosphatidylserine (DSPS), palmitoyl-oleyl-phosphatidylserine (POPS), distearoyl-phosphatidylcholine (DSPC), or C16-ceramide (CER). POPC liposomes displayed a single thermotropic transition centered at -3.4[degrees]C. When present, the second lipid formed a domain within the POPC bilayer, as indicated by the appearance of an additional peak. This peak was centered at temperatures close to 0[degrees]C in the case of liposomes containing 10% CER, DSPS, and POPS and at 23[degrees]C in the case of DSPC, likely reflecting a higher degree of molecular packing for DSPC domains. In DSPS/POPC, POPS/POPC, or CER/POPC, but not in DSPC/POPC liposomes, the insertion of [b.sub.5]wt increased, whereas [b.sub.5]ext decreased, the relative contribution to the total enthalpy of the higher temperature, phase-separated component. These results were confirmed with fluorescence measurements by using pyrene-labeled phospholipids. The dissimilar interaction with lipids of these two differently localized TA proteins could have implications for their intracellular sorting. cytochrome [b.sub.5] | differential scanning calorimetry | endoplasmic reticulum | lipid domains | liposomes

Studies undertaken in the past six years have shown that nitric oxide and many members of the sphingolipid family interact in two-way systems, whereby each 'player' regulates and is regulated by another 'player'. These interactions take place at the level of the expression and activity of several enzymes that regulate both the sphingolipid metabolic pathways and the generation of nitric oxide. So far, the outcomes of these interactions have been elucidated only in part. It is already clear, however, that they play prominent roles in key biological processes, including apoptosis, inflammation and cell growth.

The role of myristic acid in the interaction of reductase with liposomes and target membrane selection was analyzed by comparing the localization of nonmyristoylatable G2A mutant with wild-type reductase in mammalian cells. The subcellular distribution of the membrane-bound nonmyristoylatable G2A mutant was significantly altered compared to wild-type nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. However, the ability of the mutant protein to bind with the membranes of the endoplasmic reticulum was not altered by the mutation.

Many resident membrane proteins of the endoplasmic reticulum (ER) do not have known retrieval sequences. Among these are the so-called tail-anchored proteins, which are bound to membranes by a hydrophobic tail close to the C terminus and have most of their sequence as a cytosolically exposed N-terminal domain. Because ER tail-anchored proteins generally have short ([less than or equal to]17 residues) hydrophobic domains, we tested whether this feature is important for localization, using cytochrome [b.sub.5] as a model. The hydrophobic domain of cytochrome [b.sub.5] was lengthened by insertion of five amino acids (ILAAV), and the localization of the mutant was analyzed by immunofluorescence in transiently transfected mammalian cells. While the wild-type cytochrome was localized to the ER, the mutant was relocated to the surface. This relocation was not due to the specific sequence introduced, as demonstrated by the ER localization of a second mutant, in which the original length of the membrane anchor was restored, while maintaining the inserted ILAAV sequence. Experiments with brefeldin A and with cycloheximide demonstrated that the extended anchor mutant reached the plasma membrane by transport along the secretory pathway. We conclude that the short membrane anchor of cytochrome [b.sub.5] is important for its ER residency, and we discuss the relevance of this finding for other ER tail-anchored proteins.