TRAP assists membrane protein topogenesis at the mammalian ER membrane

Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them. Here we present evidence that the translocon-associated protein (TRAP) complex is involved in membrane protein topogenesis in vivo. Small interfering RNA (siRNA)-mediated silencing of the TRAP complex in HeLa cells enhanced the topology effect of mutating the flanking charges of a signal-anchor, but not of increasing signal hydrophobicity. The results suggest a role of the TRAP complex in moderating the ‘positive-inside’ rule.     

Are membrane enzymes regulated by the viscosity of the membrane environment?

We have examined the idea that membrane enzymes are regulated by the viscosity of surrounding lipids using data compiled from the literature for the effect of the change in membrane viscosity ([symbol: see text]) at the gel- to liquid-crystal-phase transition on the activities of several enzymes. The analysis was not extended explicitly to the problem of viscosity-dependent regulation of membrane enzymes in liquid-crystalline lipids because of the absence of exact data for values of [symbol: see text] in liquid-crystalline phases of variable composition. For most membrane enzymes studied, energies of activation are discontinuous, while kcat is continuous, at the main-phase transition. We consider that the energy of activation contains terms related to the height of the chemical barrier to reaction and terms due to the mechanical properties of the bilayer, such as the work of expansion during the catalytic cycle and the temperature dependence of [symbol: see text]. We find that the differences in energies of activation, above and below the break points in Arrhenius plots, are orders of magnitude larger than can be accounted for by the above mechanical factors. Thus, discontinuities in energies of activation at the phase transition appear to reflect changes in the chemical barrier to reaction, which is independent of [symbol: see text]. The theorectical analysis indicates too that values of [symbol: see text] for bilayers in the liquid-crystalline phase would have to be several orders of magnitude larger than those for gel phases in order to provide a basis for viscosity-dependent regulation of membrane enzymes in liquid-crystalline phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Procalpain is activated on the plasma membrane and the calpain acts on the membrane

The mechanism of activation of human erythrocyte calpain was investigated using the immunoblotting technique with anticalpain monoclonal antibody. The purified calpain underwent a Ca2+-induced fragmentation of the 80 kDa subunit to 76 kDa and 36 kDa fragments. The behavior of the 76 kDa fragment in electrophoresis corresponded to the proteinase activity of calpain, whereas the behavior of the 80 kDa subunit and the 36 kDa fragment did not. When inside-out membrane vesicles were added to the reaction mixture of calpain and Ca2+ and the vesicles were separated from the supernatant solution by centrifugation, the 80 kDa subunit and 76 kDa fragment were found in the vesicle fraction. No other fragments were found in this fraction. On the other hand, the 80 kDa subunit and 36 kDa fragment were found in the supernatant fraction. When right-side-out membrane vesicles were added to the reaction mixture and the vesicles were separated from the supernatant fraction, no fragment was found in the vesicle fraction, while only the 36 kDa fragment was found in the supernatant fraction. These results indicate that the 80 kDa subunit of procalpain was bound in a Ca2+-dependent manner to the cytosolic surface of the plasma membrane and then underwent fragmentation to produce the 76 kDa fragment (active form) and that it expressed its proteinase activity at the surface of the membrane.     

Structural insights into the membrane chaperones for multi-pass membrane protein biogenesis

Certain transmembrane α-helices of multi-pass membrane proteins line substrate transport paths or catalytic pockets and, therefore, are partially hydrophilic. Sec61 alone is insufficient to insert these less hydrophobic segments into the membrane and needs to work with dedicated membrane chaperones. Three such membrane chaperones have been described in the literature—the endoplasmic reticulum membrane protein complex (EMC), the TMCO1 complex, and the PAT complex. Recent structural studies on these membrane chaperones have revealed their overall architecture, multi-subunit assembly, putative substrate transmembrane helix-binding pockets, and cooperative mechanisms with the ribosome and Sec61 translocon. These structures are providing initial insights into the poorly understood processes of multi-pass membrane protein biogenesis.     

Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins

Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae. In this article, four GPI-anchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus. Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions. The phosphatidylinositol moiety of the A. fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C24:0 fatty acid. In constrast to yeast, only ceramide was found in the GPI anchor structures of A. fumigatus, even for Gel1p, a homolog of Gas1p in S. cerevisiae that contains diacylglycerol. The A. fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Manalpha1-3Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN.     

Sane in the membrane: designing systems to modulate membrane proteins

Proteins and lipids make sense in rational approaches to the design of systems for the study of membrane proteins. Lipids surround integral membrane proteins in their natural environment. Although lipids have always formed part of investigations into membrane proteins, it has generally been the proteins themselves that have taken the limelight. As knowledge of membrane proteins has increased, so has that of their interactions with lipids. This increased understanding of the interplay of proteins and lipids, together with existing knowledge of lipid properties, is enabling new approaches to be introduced for membrane protein study. The lipids can be used to control protein behaviour and as novel probes of protein motion.     

Antibodies to the ciliary membrane of Paramecium tetraurelia alter membrane excitability

Immobilization of Paramecium followed the binding of antibodies to the major proteins of the ciliary membrane (the immobilization antigens, i- antigens, approximately 250,000 mol wt). Immunoelectron microscopy showed this binding to be serotype-specific and to occur over the entire cell surface. Antibody binding also reduced the current through the Ca-channel of the excitable ciliary membrane as monitored using a voltage-clamp. The residual Ca-current appeared normal in its voltage sensitivity and kinetics. As a secondary consequence of antibody binding, the Ca-induced K-current was also reduced. The resting membrane characteristics and other activatable currents, however, were not significantly altered by the antibody treatment. Since monovalent fragments of the antibodies also reduced the current but did not immobilize the cell, the electrophysiological effects were not the secondary consequences of immobilization. Antibodies against the second most abundant family of proteins (42,000-45,000 mol wt) had similar electrophysiological effects as revealed by experiments in which the Paramecia and the serum were heterologous with respect to the i-antigen but homologous with respect to the 42,000-45,000-mol-wt proteins. Protease treatment, shown to remove the surface antigen, also caused a reduction of the Ca-inward current. The loss of the inward Ca-current does not seem to be due to a drop in the driving force for Ca++ entry since increasing the external Ca++ or reducing the internal Ca++ (through EGTA injection) did not restore the current. Here we discuss the possibilities that (a) the major proteins define the functional environment of the Ca-channel and that (b) the Ca-channel is more susceptible to certain general changes in the membrane.     

Protein translocation into and across the bacterial plasma membrane and the plant thylakoid membrane

Over the past decade, some familiar themes have emerged on how proteins are inserted into or translocated across the plant chloroplast thylakoid membrane and bacterial inner membranes. In the SecA and signal recognition particle (SRP) pathways, nucleotides and soluble factors are used to translocate proteins across the membrane bilayer in the unfolded state. However, the delta pH-dependent pathway in thylakoids uses a radically different mechanism: transport of proteins across the membrane is driven by the transmembrane pH gradient, and neither stromal factors nor nucleotide triphosphates are needed. In addition, this pathway, which requires the membrane-bound protein Hcf106, appears to translocate proteins in a tightly folded form. Recently, a similar pathway has been shown to operate in eubacteria, and several of its components have been identified.     

The importance of the membrane interface as the reference state for membrane protein stability

The insertion of nascent polypeptide chains into lipid bilayer membranes and the stability of membrane proteins crucially depend on the equilibrium partitioning of polypeptides. For this, the transfer of full sequences of amino-acid residues into the bilayer, rather than individual amino acids, must be understood. Earlier studies have revealed that the most likely reference state for partitioning very hydrophobic sequences is the membrane interface. We have used μs-scale simulations to calculate the interface-to-transmembrane partitioning free energies ΔG_S→TM for two hydrophobic carrier sequences in order to estimate the insertion free energy for all 20 amino acid residues when bonded to the center of a partitioning hydrophobic peptide. Our results show that prior single-residue scales likely overestimate the partitioning free energies of polypeptides. The correlation of ΔG_S→TM with experimental full-peptide translocon insertion data is high, suggesting an important role for the membrane interface in translocon-based insertion. The choice of carrier sequence greatly modulates the contribution of each single-residue mutation to the overall partitioning free energy. Our results demonstrate the importance of quantifying the observed full-peptide partitioning equilibrium, which is between membrane interface and transmembrane inserted, rather than combining individual water-to-membrane amino acid transfer free energies.