Transient domain formation in membrane-bound organelles undergoing maturation

The membrane components of cellular organelles have been shown to segregate into domains as the result of biochemical maturation. We propose that the dynamical competition between maturation and lateral segregation of membrane components regulates domain formation. We study a two- component fluid membrane in which enzymatic reaction irreversibly converts one component into another, and phase separation triggers the formation of transient membrane domains. The maximum domains size is shown to depend on the maturation rate as a power-law similar to the one observed for domain growth with time in the absence of maturation, despite this time dependence not being verified in the case of irreversible maturation. This control of domain size by enzymatic activity could play a critical role in intra-organelle dynamics.Comment: 7 pages, 6 figure

Metagenomic sequencing unravels gene fragments with phylogenetic signatures of O2-tolerant NiFe membrane-bound hydrogenases in lacustrine sediment

Many promising hydrogen technologies utilising hydrogenase enzymes have been slowed by the fact that most hydrogenases are extremely sensitive to O2. Within the group 1 membrane-bound NiFe hydrogenase, naturally occurring tolerant enzymes do exist, and O2 tolerance has been largely attributed to changes in iron–sulphur clusters coordinated by different numbers of cysteine residues in the enzyme’s small subunit. Indeed, previous work has provided a robust phylogenetic signature of O2 tolerance [1], which when combined with new sequencing technologies makes bio prospecting in nature a far more viable endeavour. However, making sense of such a vast diversity is still challenging and could be simplified if known species with O2-tolerant enzymes were annotated with information on metabolism and natural environments. Here, we utilised a bioinformatics approach to compare O2-tolerant and sensitive membrane-bound NiFe hydrogenases from 177 bacterial species with fully sequenced genomes for differences in their taxonomy, O2 requirements, and natural environment. Following this, we interrogated a metagenome from lacustrine surface sediment for novel hydrogenases via high-throughput shotgun DNA sequencing using the Illumina™ MiSeq platform. We found 44 new NiFe group 1 membrane-bound hydrogenase sequence fragments, five of which segregated with the tolerant group on the phylogenetic tree of the enzyme’s small subunit, and four with the large subunit, indicating de novo O2-tolerant protein sequences that could help engineer more efficient hydrogenases

Characterization of the extracellular lipase of Bacillus subtilis and its relationship to a membrane-bound lipase found in a mutant strain

Bacillus subtilis CMK33 is a mutant that is more osmotically fragile than the wild type when it is converted to the protoplast form. The protoplasts of this mutant contain a membrane-bound lipase, which is not found in protoplasts of the wild type. Hydrolysis of the membrane lipid of mutant protoplasts by the lipase is the cause of their fragility. A protein found in the wild type organism specifically inhibits the lipase (Kent, C., and Lennarz, W. J. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2793-2797). This paper reports that cultures of both mutant and wild type cells contain an extracellular lipase which accumulates during the logarithmic phase of growth. The extracellular activity appears to be induced by a component of the growth medium. The membrane-bound lipase of the mutant has been partially purified and its properties have been compared to those of the extracellular lipase of the wild type. Their properties and sensitivity to the wild type inhibitor are similar, which suggests that the two molecules are closely related. The subcellular location of the lipase in the mutant has been investigated and compared to the location of the membrane-bound portion of the lipase inhibitor in the wild type. The lipase is located almost exclusively in the cytoplasmic membrane and not in mesosomal vesicles. In contrast, the lipase inhibitor is located in both types of membranes and is more concentrated in mesosomal vesicles. Under appropriate conditions, the appearance of new extracellular lipase activity in mutant cultures is paralleled by the loss of an equivalent amount of lipase activity from protoplasts prepared from the cells. This suggests that the membrane-bound lipase may be an intermediate in the secretion of the extracellular lipase. Because of the mutation in B. subtilis CMK33, which results in the absence of the lipase inhibitor, this intermediate can be found in protoplasts of the mutant, although it is not detectable in the wild type. Consequently, the mutant may be useful in studies of the mechanism of secretion of exoenzymes by Bacilli

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as well as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic Percoll™ gradient. In this way, secretory vesicles were separated from mitochondrial, microsomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 μmol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg2+ and Ca2+ concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonic media (<400 mosmol/kg) or in Triton X-100 (0.2% final concentration) acetylcholinesterase activity was markedly higher. During hypotonic treatment or when secretory vesicles were specifically lyzed with 2 mM Mg2+ and 2 mM ATP, adrenalin as well as part of acetylcholinesterase was released from the vesicular content. On polyacrylamide gel electrophoresis this soluble enzyme exhibited the same electrophoretic mobility as the enzyme released into the perfusate from adrenal glands upon stimulation. In addition to the soluble enzyme a membrane bound form of acetylcholinesterase exists within secretory vesicles, which sediments with the secretory vesicle membranes and exhibits a different electrophoretic mobility compared to the soluble enzyme. It is concluded, that the soluble enzyme found within isolated secretory vesicles is secreted via exocytosis, whilst the membrane-bound form is transported to the cell membrane during this process, contributing to the biogenesis of the cell membrane

Immunity to MHC class I antigen after direct DNA transfer into skeletal muscle.

Plasmid cDNA encoding the alpha-chain of either membrane-bound (pcRT.45) or secreted (pcRQ.B3) RT1Aa MHC class I Ag were transferred to Lewis (RT1(1)) rat skeletal muscle by direct injection. Rats were challenged 7 days later with an ACI (RT1a) heterotropic heart transplant, and cardiac allograft survival, RT1Aa-specific antibody levels, and frequency of ACI-specific CTL were monitored. Graft rejection was accelerated by > or = 2 days in an Ag-specific and dose-dependent manner in pcRT.45-injected rats. The pcRQ.B3-injected rats also rejected grafts more rapidly; however, graft rejection was accelerated by only 1 day, and graft infiltrates were less pronounced than in pcRT.45-injected rats. Injection of pcRT.45 resulted in an increase in ACI-specific CTL precursor frequency 3 days post-transplant, whereas there was no significant change in rats pretreated with pcRQ.B3 injection. Compared with rats injected with a control plasmid encoding firefly luciferase, transfer of pcRT.45 resulted in an increase in RT1Aa-specific IgG and IgM antibody 3 days after heart transplantation. Transfer of pcRQ.B3 resulted in a similar mean increase in RT1Aa-specific IgG and IgM antibody after transplantation, but the variability from rat to rat was greater, with some animals exhibiting strong priming, and others showing little or no priming by gene injection. Our results suggest that skeletal muscle can express either membrane-bound or secreted MHC class I Ag after gene transfer, but that the membrane-bound form is more immunogenic than the secreted form in the high responder Lewis rat. Direct DNA transfer to skeletal muscle provides a rapid and specific approach to studying immunity to allogeneic MHC Ag

Structural Metrics Predict Site-Specific Evolutionary Rate in Membrane Bound Proteins

Membrane proteins are involved in many critical biological processes and mutations are linked to various diseases. We examined how the properties of the location of an amino acid residue within the protein structure dictates the rate at which it evolves. We tested 3 structural metrics: WCNSC, WCNCA and RSA for their effectiveness at predicting evolutionary rates within membrane proteins. WCNSC performed better than WCNCA in almost all cases and better than RSA in the majority. However, for some classes of proteins, especially those where the pore is a major feature, the effectiveness of WCN greatly diminished while that of RSA decreased by a lesser degree.Integrative Biolog

Microsatellite instability, KRAS mutations and cellular distribution of TRAIL-receptors in early stage colorectal cancer.

Thus, we evaluated the immunofluorescence pattern of TRAIL-receptors and E-cadherin to assess the fraction of membrane-bound TRAIL-receptors in 231 selected patients with early-stage CRC undergoing surgical treatment only. Moreover, we investigated whether membrane staining for TRAIL-receptors as well as the presence of KRAS mutations or of microsatellite instability (MSI) had an effect on survival and thus a prognostic effect. The fact that the receptors for the TNF-related apoptosis inducing ligand (TRAIL) are almost invariably expressed in colorectal cancer (CRC) represents the rationale for the employment of TRAIL-receptors targeting compounds for the therapy of patients affected by this tumor. Yet, first reports on the use of these bioactive agents provided disappointing results. We therefore hypothesized that loss of membrane-bound TRAIL-R might be a feature of some CRC and that the evaluation of membrane staining rather than that of the overall expression of TRAIL-R might predict the response to TRAIL-R targeting compounds in this tumor. As expected, almost all CRC samples stained positive for TRAIL-R1 and 2. Instead, membrane staining for these receptors was positive in only 71% and 16% of samples respectively. No correlation between KRAS mutation status or MSI-phenotype and prognosis could be detected. TRAIL-R1 staining intensity correlated with survival in univariate analysis, but only membranous staining of TRAIL-R1 and TRAIL-R2 on cell membranes was an independent predictor of survival (cox multivariate analysis: TRAIL-R1: p = 0.019, RR 2.06[1.12-3.77]; TRAIL-R2: p = 0.033, RR 3.63[1.11-11.84]). In contrast to the current assumptions, loss of membrane staining for TRAIL-receptors is a common feature of early stage CRC which supersedes the prognostic significance of their staining intensity. Failure to achieve therapeutic effects in recent clinical trials using TRAIL-receptors targeting compounds might be due to insufficient selection of patients bearing tumors with membrane-bound TRAIL-receptors

Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori-infected gastric cancer cells.

β-catenin has two different cellular functions: intercellular adhesion and transcriptional activity. The E3 ubiquitin ligase Siah1 causes ubiquitin-mediated degradation of the cytosolic β-catenin and therefore, impairs nuclear translocation and oncogenic function of β-catenin. However, the effect of Siah1 on the cell membrane bound β-catenin has not been studied. In this study, we identified that the carcinogenic bacterium H. pylori increased ETS2 transcription factor-mediated Siah1 protein expression in gastric cancer cells (GCCs) MKN45, AGS and Kato III. Siah1 protein level was also noticeably higher in gastric adenocarcinoma biopsy samples as compared to non-cancerous gastric epithelia. Siah1 knockdown significantly decreased invasiveness and migration of H. pylori-infected GCCs. Although, Siah1 could not increase degradation of the cytosolic β-catenin and its nuclear translocation, it enhanced degradation of the membrane-bound β-catenin in the infected GCCs. This loss of membrane-bound pool of β-catenin was not associated with the proteasomal degradation of E-cadherin. Thus, this work delineated the role of Siah1 in increasing invasiveness of H. pylori-infected GCCs