Palmitoylation is required for the activities of several cancer-associated proteins, making the palmitoyl acyltransferase (PAT) enzymes that catalyze these reactions potential targets for anticancer therapeutics. In this study, we sought to identify and characterize a human PAT with activity toward N-terminally myristoylated and palmitoylated proteins. NIH/3t3 cells were stably transfected with vectors containing no insert, wild type human DHHC20, or a serine-substituted DHHS20 mutant. Compared with control cells, cells overexpressing wild-type DHHC20 displayed an increase in palmitoylation activity toward a peptide that mimics the N-terminus of myristoylated and palmitoylated proteins, but had no change in activity toward a peptide that mimics the C-terminus of farnesylated and palmitoylated proteins. Cells expressing DHHS20 had no significant change in activity toward either peptide. Overexpression of DHHC20 also caused phenotypic changes consistent with cellular transformation, including colony formation in soft agar, decreased contact inhibition of growth, and increased proliferation under low-serum conditions. Quantitative polymerase chain reaction analyses of human tissues demonstrated that DHHC20 is expressed in a tissue-specific manner, and is overexpressed in several types of human tumors, including ovarian, breast and prostate. Overall, these results demonstrate that DHHC20 is a human N-terminal-myristoyl-directed PAT involved in cellular transformation, that may play a role in cancer.

The Hepatitis C virus (HCV) NS4B protein, a multispanning endoplasmic reticulum (ER) membrane protein, generates intracellular rearrangements of ER-derived membranes, essential for HCV replication. In this study, we characterized NS4B elements involved in the process of targeting, association and retention in the ER membrane. We investigated the localization and membrane association of a number of C- or N-terminal NS4B deletions expressed as GFP chimeras by biochemical and fluorescence microscopy techniques. A second set of GFP-NS4B chimeras containing the plasma membrane ecto-ATPase CD39 at the C-terminus of each NS4B deletion mutant was used to further examine the role of N-terminal NS4B sequences in ER retention. Several structural elements, besides the first two transmembrane domains (TMs), within the NS4B N-terminal half (residues 1-130) were found to mediate association of the NS4B-GFP chimeras with ER membranes. Both TM1 and TM2 are required for ER anchoring and retention but are not sufficient for ER retention. Sequences upstream of TM1 are also required. These include two putative amphipathic alpha-helices and a Leucine Rich Repeat-like motif, a sequence highly conserved in all HCV genotypes. The N-terminal 55peptidic sequence, containing the 1st amphipathic helix, mediates association of the 55N-GFP chimera with cellular membranes including the ER, but is dispensable for ER targeting of the entire NS4B molecule. Importantly, the C-terminal 70peptidic sequence can associate with membranes positive for ER markers in the absence of any predicted TMs. In conclusion, HCV NS4B targeting and retention in the ER results from the concerted action of several NS4B structural elements.

The attachment of palmitic acid to the amino acid cysteine via thioester linkage (S-palmitoylation) is a common post-translational modification of eukaryotic proteins. In this review, we discuss the role of palmitoylation as a versatile protein sorting signal, regulating protein trafficking between distinct intracellular compartments and the micro-localization of proteins within membranes.

Adrenergic receptors are integral membrane proteins involved in cellular signalling that belong to the G protein-coupled receptors. Synthetic peptides resembling the putative transmembrane (TM) segments TM4, TM6 and TM7, of the human alpha2-adrenergic receptor subtype C10 (P08913) and defined lipid vesicles were used to assess protein-lipid interactions that might be relevant to receptor structure/function. P6 peptide contains the hydrophobic core of TM6 plus the N-terminal hydrophilic motif REKR, while peptides P4 and P7 contained just the hydrophobic stretches of TM4 and TM7, respectively. All the peptides increase their helical tendency at moderate concentrations of TFE (30-50%) and in presence of 1,2-dielaidoyl-sn-glycero-3-phosphatidylethanolamine (DEPE) lipids. However, only P6 displays up to 19% of alpha-helix in the presence of just the DEPE lipids, evidences a transmembrane orientation and stabilizes the Lalpha lipid phase. Conversely, P4 and P7 peptides form only stable beta-sheet structures in DEPE and favour the non-lamellar, inverted hexagonal (H(II)) phase of DEPE by lowering its phase transition temperature. This study highlights the potential of using synthetic peptides derived from the amino acid sequence in the native proteins as templates to understand the behaviour of the transmembrane segments and underline the importance of interfacial anchoring interactions to meet hydrophobic matching requirements and define membrane organization.

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8+/+, Slc2a8+/−, and Slc2a8−/− mice, respectively) resulting in a deviation (p = 0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.

The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate > phosphatidylserine > phosphatidylcholine approximately = phosphatidylethanolamine approximately = 0. Acidic phospholipids increased V(max-Ca2+) and lowered the value of K(0.5-Ca2+) below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K(0.5-Ca2+) value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1-116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Delta74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca2+-ATPase provides new evidence that type 2B Ca2+-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.

The role of the side chain of sterols and the sterol ring structure on the formation of ordered phases of the type observed in membrane rafts has been examined in aqueous dispersions of binary mixtures of sphingomyelin and androsterol. Comparisons have been made with binary systems of cholesterol, stigmasterol, beta-sitosterol, and ergosterol with either sphingomyelin or dipalmitoylphosphatidylcholine. Thermotropic phase behaviour and structure of the mixed aqueous dispersions were characterized by differential scanning calorimetry, synchrotron X-ray diffraction, freeze-fracture electron microscopy, and Fourier-transform infrared spectroscopy. We show that: (i) Androsterol is less efficient in promoting the formation of liquid-ordered phase than other naturally occurring sterols which possess a side chain, (ii) cholesterol is the most efficient sterol of those investigated in forming liquid-ordered phase, (iii) the molecular stoichiometry of egg sphingomyelin and androsterol in the liquid-ordered phase is about 2:1, and (iv) sphingomyelin can form more stable liquid-ordered phase than glycerophospholipid in binary systems containing androsterol.

Mediation of synchronous cell-cell interactions by NCAM and PSA-NCAM is examined here in aggregates (monolayers) of C6 polysialylated embryonic neural cells, formed rapidly (within 30 s) in suspension in an ultrasound trap. These cells express all three main isoforms of neural cell adhesion molecule (NCAM). The rate of extension of perimeter contact (i.e., membrane spreading) between closely adjacent cells and the temporal reinforcement of the Filamentous (F)-actin cytoskeleton at those regions were measured. Enzymatic removal of the cell-cell repelling polysialic acid (PSA) increases the rate of NCAM-induced membrane spreading, while removal of NCAM-120 had no detectable effect. Competitive peptide inhibition of the third immunoglobulin domain of NCAM significantly reduced the rate of membrane spreading, while NCAM siRNA transfected cells lost their ability to spread. It is argued that NCAM induced contact is the initial requirement for membrane spreading and facilitates conditions for subsequent cytoskeletal reorganization in these neural cells.

Aminopeptidase N (APN/CD13) is a 150 kDa membrane-bound ubiquitously expressed protease with a broad functional repertoire. It hydrolyzes small peptide mediators, modulates cell motility and adhesion to extracellular matrix and also acts as a viral receptor. In order to dissect the function of enzymatically active and inactive APN/CD13, substitutions of different enzymatic active amino acid residues were generated by site-directed mutagenesis and stably transfected into human embryonic kidney cells. All APN variants analyzed exhibited a complete loss of enzymatic activity, whereas wild type APN transfectants exerted a strong aminopeptidase-specific activity. Furthermore, wild type APN expression was associated with a significant decrease in proliferation, migration and also reduced anchorage-independent growth when compared to enzymatically inactive APN variants and controls. This appeared to be due to a downregulated mRNA and protein expression of the chemokine receptor CXCR4 and an inhibition of the stromal cell-derived factor (SDF)-1alpha/CXCL12-mediated migration. Thus, high APN enzyme activity may antagonize the cellular properties regulated by the CXCR4/SDF-1alpha system in embryonic kidney cells.

Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidth analysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 to I298, indicating that it has an alpha-helical structure. Saturation behaviour, in presence of paramagnetic perturbants of different hydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect to the lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activity data indicate that the segment is made by an alpha helix, accommodated in an amphipathic environment, partially distorted in the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region of the segment is less restrained and more flexible than the N-terminus.