Membrane Polypeptides Research Articles

Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy

Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the (15)N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the (2)H solid-state NMR spectra acquired from peptides labelled with 3,3,3-(2)H(3)-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the C(alpha)-C(beta) bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu(1-27) and Influenza A M2(22-46) are shown. Whereas the (15)N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed.

Photosynthetic and biochemical activities in flag leaves of a newly developed superhigh-yield hybrid rice (Oryza sativa) and its parents during the reproductive stage

Responses of net photosynthetic rates to intercellular CO(2) concentration (P (n)/C (i) curves) and photochemical characteristics were investigated in flag leaves of newly developed superhigh-yield hybrid rice (Oryza sativa L.) LiangYouPeiJiu (LYPJ) and its maternal PeiAi64S (PA64S) and paternal WuMang9311 (WM9311) lines grown in the field during the reproductive stage. The results showed that photosynthetic functions, such as the electron transport activities of photosystems and photophosphorylation, assessed in vivo from P (n)/C (i) curves under field conditions declined more or earlier than those obtained in vitro. The degradation of polypeptides of thylakoid membranes was slower than those for P (Ca=360) (light-saturated net photosynthetic rate measured at 360 mumol mol(-1)) and CE (carboxylation efficiency, obtained from the initial slope of the P (n)/C (i) curve). The initial inhibition of the PSII electron transport and oxygen-evolving activity induced by senescence occurred before the degradation of the oxygen-evolving complex. In comparison, LYPJ had intermediate photosynthetic functions in the early stage of leaf development, but greater photochemical activities in the mid and late stages. WM9311 showed a similar pattern of changes but lower values, and PA64S had higher values in the early stage but showed a faster rate of senescence than LYPJ. These findings implied that the hybrid LYPJ demonstrated intermediate photosynthetic activities between its parents in the early stage of leaf development, whereas it had higher photosynthetic activities than its parents in the mid and late stages, which may be responsible for its high yield.

Purification of the NF2 Tumor Suppressor Protein from Human Erythrocytes

Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane. Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a approximately 70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins. These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately 41-65,000 molecules of NF2 protein per erythrocyte. We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.

PsbI Affects the Stability, Function, and Phosphorylation Patterns of Photosystem II Assemblies in Tobacco

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.

Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.

Functional roles of the gamma subunit of the skeletal muscle DHP-receptor

In excitation-contraction coupling (EC coupling) of skeletal muscle, large and rapid changes of the myoplasmic Ca2+ concentration mediate the activation and termination of force. The L-type Ca2+ channel (dihydropyridine receptor, DHP receptor) is a central component of the EC coupling process. Its predominant role is to provide the Ca2+ release channels of the sarcoplasmic reticulum (SR) with the sensitivity to cell membrane voltage. The DHP receptor consists of five different proteins (alpha1S, beta1, gamma1, delta and alpha2) whose tasks and functional characteristics are still incompletely understood. This short review summarizes progress made in studying the physiology of the gamma1 subunit, a membrane polypeptide that is highly specific for skeletal muscle. The focus is on recent results obtained from muscle of gamma1-deficient mice.

Cloning and characterization of corA, a gene encoding a copper-repressible polypeptide in the type I methanotroph, Methylomicrobium albus BG8.

In an attempt to identify proteins involved in copper transport in the type I methanotroph Methylomicrobium albus BG8, copper-regulated polypeptides were examined. One major copper-repressible membrane polypeptide of approx. 28,500 Da was identified and designated CorA. The gene encoding this polypeptide was isolated and sequenced, and it shared a low identity with a calcium channel protein. An insertion mutation in corA of M. albus BG8 grew very poorly, suggesting that CorA is important for growth of this methanotroph. CorA may be involved in transport of copper and/or other divalent metals ions in M. albus BG8.

Protein−Protein Interactions: Modeling the Hepatitis C Virus Ion Channel p7

The p7 protein is a small ion-channel-forming membrane polypeptide encoded by the hepatitis C virus which consists of two transmembrane alpha-helices, TM1 and TM2, and can be blocked by long-alkyl-chain iminosugar derivatives. The length of TM1 and TM2 was estimated by employing different secondary structure prediction algorithms and is proposed to span from Ala-10 to Leu-32 for TM1 and from Trp-36 to Pro-58 for TM2. A configurational search protocol based on simulated annealing combined with short restrained molecular dynamics simulations is used in addition to protein-protein docking to investigate the packing of TM1/TM2. Full p7 oligomeric bundles were generated, and in the most plausible models serines and threonines are facing the hydrophilic pore. In these models, His-17 would be a pore-facing residue, suggesting that p7 may be sensitive to pH in respect to its function.

Yos9p Detects and Targets Misfolded Glycoproteins for ER-Associated Degradation

Endoplasmic reticulum (ER) quality control mechanisms monitor the folding of nascent secretory and membrane polypeptides. Immature molecules are actively retained in the folding compartment whereas proteins that fail to fold are diverted to proteasome-dependent degradation pathways. We report that a key pathway of ER quality control consists of a two-lectin receptor system consisting of Yos9p and Htm1/Mnl1p that recognizes N-linked glycan signals embedded in substrates. This pathway recognizes lumenally oriented determinants of soluble and membrane proteins. Yos9p binds directly to substrates to discriminate misfolded from folded proteins. Substrates displaying cytosolic determinants can be degraded independently of this system. Our studies show that mechanistically divergent systems collaborate to guard against passage and accumulation of misfolded proteins in the secretory pathway.

Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase

The H2O2 byproduct of fatty acid catabolism in plant peroxisomes is removed in part by a membrane-associated antioxidant system that involves both an ascorbate peroxidase and a monodehydroascorbate reductase (MDAR). Despite descriptions of 32-kDa MDAR polypeptides in pea and castor peroxisomal membranes and cDNA sequences for several 'cytosolic' MDARs, the genetic and protein factors responsible for peroxisomal MDAR function have yet to be elucidated. Of the six MDAR polypeptides in the Arabidopsis proteome, named AtMDAR1 to AtMDAR6 in this study, 47-kDa AtMDAR1 and 54-kDa AtMDAR4 possess amino acid sequences that resemble matrix (PTS1) and membrane peroxisomal targeting signals, respectively. Epitope-tagged versions of these two MDARs and a pea 47-kDa MDAR (PsMDAR) sorted in vivo directly from the cytosol to peroxisomes in Arabidopsis and BY-2 suspension cells, whereas AtMDAR2 and AtMDAR3 accumulated in the cytosol. The PTS1-dependent sorting of AtMDAR1 and PsMDAR to peroxisomes was incomplete (inefficient?), but was improved for PsMDAR after changing its PTS1 sequence from -SKI to the canonical tripeptide -SKL. A C-terminal transmembrane domain and basic cluster of AtMDAR4 were necessary and sufficient for targeting directly to peroxisomes. MDAR activity in isolated Arabidopsis peroxisomes was distributed among both water-soluble matrix and KCl-insoluble membrane subfractions that contained respectively 47- and 54-kDa MDAR polypeptides. Notably, a 32-kDa MDAR was not identified. Combined with membrane association and topological orientation findings, these results indicate that ascorbate recycling in Arabidopsis (and probably other plant) peroxisomes is coordinated through functionally redundant MDARs that reside in the membrane and the matrix of the organelle.

Putative mineral-specific proteins synthesized by a metal reducing bacterium

Biological force microscopy (BFM) was combined with two-dimensional (2D) gel electrophoresis and mass spectrometry to identify outer membrane proteins (OM) from Shewanella oneidensis that are involved in anaerobic Fe(III) reduction. This is the first time that biophysical force measurements have been coupled with protein expression patterns to search for evidence of putative mineral-specific proteins synthesized by bacteria. BFM shows that S. oneidensis possess an affinity towards goethite (FeOOH) but not diaspore (AlOOH) under anaerobic conditions, despite the fact that diaspore is isostructural with goethite and has essentially the same surface charge. The worm-like chain model was used to identify force-signatures in BFM-derived force curves indicative of putative outer membrane (OM) polypeptides synthesized by S. oneidensis to form a bond with goethite. Protein expression patterns from OM extract of cells grown under anaerobic Fe(III) reducing versus aerobic conditions show that approximately 400 protein spots exhibit significant differences in abundance on 2D gels. Peptide mass fingerprinting and tandem mass spectrometry were used to identify several of the protein spots that were significantly more abundant in 2D gels from OM extract of cells grown under anaerobic Fe(III) reducing conditions. Among those identified were proteins involved in Fe(III) and Mn(IV) reduction, protein transport andmore » secretion, polysaccharide biosynthesis and export, and hypothetical proteins with unknown functions. Together, the BFM and proteomic data suggest that OM proteins are synthesized by S. oneidensis under anaerobic conditions to function in iron oxide binding and/or Fe(III) reduction. If this is the case, then it is possible that the evolution of dissimilatory iron-reducing bacteria like Shewanella, could have been, at least in part, driven by the binding/reduction ability of certain proteins to specific mineral phases.« less

Magic angle spinning and static oriented sample NMR studies of the relaxation in the rotating frame of membrane peptides

To demonstrate the influence of motions with medium-to-slow correlation times (milliseconds to nanoseconds), a systematic study of the spin-lattice relaxation in the rotating frame was conducted for several nuclei (H1, C13, and N15) in small membrane polypeptides, either in oriented or magic angle spinning samples. This study not only assesses the validity of some motional models, but also characterizes the magnetization relaxation rates crucial for the design of polarization transfer experiments. It was found that relaxation time constant (T1ρ) values on the order of 10−3–10−2s for backbone nuclei and their dependence on sample orientation are consistent with the model of transmembrane polypeptides undergoing axial diffusion (τc∼10−8–10−7s) and small amplitude off-axis reorientation (τc∼10−6–10−5s).

Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells

H(+) transport in the collecting duct is regulated by exocytic insertion of H(+)-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H(+)-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H(+)-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H(+)-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1ADeltaC [amino acids (aa) 1-264] formed complexes with H(+)-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H(+)-ATPase (aa 235-264) and another that bound SNAP23 and VAMP (aa 190-234) to an equivalent degree as full-length syntaxin. However, the aa 235-264 cassette alone without the SNARE N (aa 1-160) does not bind but requires ligation to the SNARE N to bind H(+)-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H(+)-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H(+)-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H(+)-ATPase exocytosis.

Effector-induced Syk-mediated phosphorylation in human erythrocytes

Band 3 (AE1), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72 syk) and Lyn (p53/56 lyn). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane.

Topological analysis of integral membrane constituents of prokaryotic ABC efflux systems

The ATP binding cassette (ABC) superfamily consists of dozens of families of transport systems, each of which catalyzes uptake or efflux of a specific type of molecule using ATP hydrolysis to energize transport. While all of the ATP hydrolyzing subunits in the superfamily are homologous, a monophyletic origin of the integral membrane constituents is not established. We have identified a subset of these transmembrane proteins that have a basal unit of four transmembrane α-helical segments (TMSs) with a large extracytoplasmic domain between TMSs 1 and 2. These homologues were found to exhibit 4, 8 or 10 putative TMSs per polypeptide chain. The two larger topological types exhibit a 4 TMS repeat element resulting from an internal gene duplication event, and the 10 TMS proteins display an extra two putative TMSs between the two repeat units. Rare intragenic deletions in these homologues gave rise to truncated proteins lacking the extracytoplasmic domain, and some phylogenetic clusters of the 4 TMS membrane proteins (but not the 8 or 10 TMS proteins) are fused N-terminal (never C-terminal) to ATP hydrolyzing domains. Bioinformatic analyses lead to the suggestion that in the larger homologues, the second repeat units are more important for function than the first repeat units. Operon analyses suggest that the 4 TMS proteins form heterodimeric complexes while the 8 and 10 TMS proteins incorporate the equivalent of these complexes into single integral membrane polypeptide chains. Different gene compositions of the operons encoding the 4 versus 8 and 10 TMS homologues suggest that these two structural types of transporters act on different types of substrates and serve dissimilar functions. Significant sequence similarity between the integral membrane constituents of the ABC efflux pumps analyzed here and those of other ABC transporters could not be detected. These studies define the evolutionary pathway taken for the appearance of a subset of ABC transmembrane transport proteins and provide clues regarding their mechanistic and functional characteristics.

Cell density-dependent gene contributes to efficient seed colonization by Pseudomonas putida KT2440.

We have characterized the expression pattern of a gene, ddcA, involved in initial colonization of corn seeds by Pseudomonas putida KT2440. The ddcA gene codes for a putative membrane polypeptide belonging to a family of conserved proteins of unknown function. Members of this family are widespread among prokaryotes and include the products of a Salmonella enterica serovar Typhimurium gene expressed during invasion of macrophages and psiE, an Escherichia coli phosphate starvation-inducible gene. Although its specific role is undetermined, the presence of ddcA in multicopy restored the seed adhesion capacity of a KT2440 ddcA mutant. Expression of ddcA is growth phase regulated, being maximal at the beginning of stationary phase. It is independent of RpoS, nutrient depletion, or phosphate starvation, and it is not the result of changes in the medium pH during growth. Expression of ddcA is directly dependent on cell density, being also stimulated by the addition of conditioned medium and of seed exudates. This is the first evidence suggesting the existence of a quorum-sensing system in P. putida KT2440. The potential implication of such a signaling process in seed adhesion and colonization by the bacterium is discussed.

Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing.

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient. Consecutive sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that both matrix and the integral membrane proteins of peroxisomes could be successfully fractionated and then identified by mass spectrometry. This is documented by the detection of PMP22, which is the most hydrophobic and basic protein of the peroxisomal membrane with a pI > 10. The identification of 96 prominent spots corresponding to polypeptides with different physical and chemical properties, e.g., the most abundant integral membrane polypeptides of peroxisomes and specific ones of the mitochondrial and microsomal membrane, reflects the fractionation potential of free-flow (FF)-IEF, accentuating its value in proteomic research as an alternative perhaps superior to gel-based IEF.

Native chemical ligation of hydrophobic [corrected] peptides in lipid bilayer systems.

The covalent modification of water-insoluble membrane polypeptides incorporated into lipid bilayers by native chemical ligation is described. The key feature of this strategy is the use of cubic lipidic phase (CLP) matrixes as reaction media. The CLP-matrix consists of a lipid bilayer into which hydrophobic polypeptides and folded membrane proteins can be inserted and two unbounded aqueous channels that give the aqueous phase access to both sides of an infinite lipid bilayer and thus ensure that modification of solvent-exposed sites is independent of the topology of membrane incorporation. The enzymatic removal of an N-terminal proteolytic cleavage sequence from the membrane polypeptide exposes an N-terminal cysteine residue. Subsequently, a C-terminal thioester peptide is joined to the N-terminus of the polypeptide by a native chemical ligation reaction. By use of this approach, incorporation of a variety of molecular tools, such as spectroscopic probes, unnatural amino acids, and molecular markers into membrane proteins that cannot be easily solubilized in detergent or denaturant solutions, may be achieved.

The glycosome membrane of Trypanosoma cruzi epimastigotes: protein and lipid composition

Highly purified glycosomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and isopycnic ultracentrifugation. Glycosomal membranes, produced by carbonate treatment of purified glycosomes, exhibited about eight main protein bands and eight minor ones. Essentially the same protein pattern was observed in the detergent-rich fraction of a Triton X-114 fractionation of whole glycosomes, indicating that most of the membrane-bound polypeptides were highly hydrophobic. The orientation of these proteins was studied by in situ labelling followed by limited pronase hydrolysis of intact glycosomes. Three glycosome membrane proteins were characterized as peripheral by comparing the protein bands patterns of membrane fractions obtained by different treatments. Noteworthy membrane polypeptides were: (1) a peripheral 75 kDa membrane protein, oriented towards the cytosol, which was the most abundant glycosomal membrane protein in exponentially growing epimastigotes but was essentially absent in stationary phase cells; (2) a pair of integral membrane proteins with molecular masses in the range of 85–100 kDa, which were only present in stationary phase cells; (3) a heme-containing 36 kDa protein, strongly associated to the membrane, present in both growth phases; (4) a very immunogenic 41 kDa integral membrane polypeptide, oriented towards the cytosol. The lipid composition of the glycosomal membranes was also investigated. The distribution of phospholipid species in glycosomes and glycosomal membranes was very similar to that of whole cells, with phosphatidyl-ethanolamine, phosphatidyl-choline, and phosphatidyl-serine as main components and smaller proportions of sphingomyelin and with phosphatidyl-inositol. On the other hand, glycosomes were enriched in endogenous sterols (ergosterol, 24-ethyl-5,7,22-cholesta-trien-3β-ol), and precursors, when compared with whole cells, a finding consistent with the proposal that these organelles are involved in the de novo biosynthesis of sterols in trypanosomatids. Index Descriptors and Abbreviations: glycosome, glycosome membrane proteins, Trypanosoma cruzi, epimastigote, phospholipids, neutral lipids, sterols; LIT, liver infusion-tryptose; TMBZ, 3,3 ′,5,5 ′-tetramethylbenzamidine; GMPs, glycosome membrane proteins; HK, hexokinase, EC 2.7.1.1; PGI, phosphoglucose isomerase, EC 5.3.1.9; MDH, malate dehydrogenase, EC 1.1.1.37; GDH, α-glycerol-3-phosphate dehydrogenase, EC 1.1.1.8; PK, pyruvate kinase, EC 2.7.1.40; PEPCK, phosphoenolpyruvate carboxykinase, EC 4.1.1.4.9; PGK, 3-phosphoglycerate kinase, EC 2.7.2.3; PPDK, phosphate pyruvate dikinase, EC 2.7.9.1; NL, neutral lipids; PL, phospholipids; TCA, trichloracetic acid

Hydrophobic pockets at the membrane interface: an original mechanism for membrane protein interactions.

The effect of partial digestion by trypsin and GluC protease on the association of the membrane polypeptides of LH1 from Rhodospirillum (Rsp.) rubrum was studied. Trypsin and GluC protease treatments of LH1 result in the cleavage of the first three amino acids from the alpha polypeptide and of the first 18 amino acids from the beta polypeptide, respectively, without any noticeable reorganization of their secondary structure, as measured by attenuated total reflectance Fourier transform IR spectroscopy. However, the enthalpy variation accompanying dimer formation was dramatically reduced by the protease attacks by as much as 80%. Our results show that the alphabeta heterodimer is mainly stabilized by hydrophobic interactions which involve the amino-terminal extensions of the participating polypeptides. Using the close homology between the polypeptides of Rsp. rubrum LH1 and that of Rsp. molischianum LH2, whose structure is known, a structural model for these "hydrophobic pockets" lying close to the membrane interface is proposed.