Predicting the topology of eukaryotic membrane proteins

We show that the so-called 'positive inside' rule, i.e. the observation that positively charged amino acids tend to be more prevalent in cytoplasmic than in extra-cytoplasmic segments in transmembrane proteins [von Heijne, G. (1986) EMBO J. 5, 3021-3027], seems to hold for all polar segments in multi-spanning eukaryotic membrane proteins irrespective of their position in the sequence and hence can be used in conjunction with hydrophobicity analysis to predict their transmembrane topology. Further, as suggested by others, we confirm that the net charge difference across the first transmembrane segment correlates well with its orientation [Hartmann, E., Rapoport, T. A. and Lodish H. F. (1989) Proc. Natl Acad. Sci. USA 86, 5786-5790], and that the overall amino-acid composition of long polar segments can also be used to predict their cytoplasmic or extra-cytoplasmic location [Nakashima, H. and Nishikawa, K. (1992) FEBS Lett. 303, 141-146]. We present an approach to the topology prediction problem for eukaryotic membrane proteins based on a combination of these methods.     

Biogenesis of purple membrane: regulation of bacterio-opsin synthesis

Thermography is a simple, noninvasive outpatient procedure which can be performed on patients with clinical symptoms of a herniated disk. A positive thermogram usually indicates that an abnormality will be found by myelography. However, the myelographic findings cannot be predicted by a negative lumbar thermogram.     

Stabilization of integral membrane proteins in aqueous solution using fluorinated surfactants

Surfactants carrying either a hydrocarbon or a fluorocarbon alkyl chain have been synthesized. The polar head was either tris(hydroxymethyl)acrylamidomethane (THAM), telomerized THAM, or a glycosylated THAM moiety. The aqueous solubility of some of these molecules was increased by oxidizing to a sulfoxide the thioether function that associates their hydrophobic and hydrophilic moieties. In all cases, the critical micellar concentration was principally determined by the length and chemical nature of the alkyl chain. The usefulness of these surfactants in handling integral membrane proteins in solution has been examined using as test materials chloroplast thylakoid membranes and the photosynthetic complex cytochrome b6f. In keeping with earlier observations in other systems, none of the fluorinated surfactants was able to solubilize thylakoid membranes. Transfer to a solution of fluorinated surfactant of b6f complexes that had been solubilized and purified in the presence of a classical detergent usually resulted in aggregation and precipitation of the protein, while most homologous molecules with hydrocarbon chains did keep the b6f complex soluble. Two of the fluorinated surfactants, however, proved able to maintain the b6f complex water-soluble, intact, and enzymatically active. Because of their limited affinity for lipid alkyl chains and other hydrocarbon surfaces, fluorinated surfactants appear as potentially interesting tools for the study of membrane proteins that do not stand well exposure to classical detergents.     

Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms

The aim of this study was to study innervation and angiogenesis in response to grafts of dermis and cultured keratinocytes using immunohistochemical techniques. In a porcine model, fresh autologous de-epidermalized dermis and cultured autologous keratinocytes were combined using a two-stage technique, to produce keratodermal grafts. Wounds were encased within skin graft chambers that prevented the influence of the surrounding skin. As grafts contracted, a peripheral rim of granulation tissue became exposed, allowing us to compare the wound bed beneath grafts with that beneath the raw granulating surface. Grafts were studied for 6 weeks. Angiogenesis was studied using antisera to von Willebrand factor to detect endothelial cells. Nerve growth was studied using antisera to S-100, a Schwann cell marker, and to four axonal markers: protein gene product 9.5, C-flanking peptide of neuropeptide Y, calcitonin gene-related peptide, and vasoactive intestinal peptide. In kerato-dermal grafts (n = 28), organization of blood vessels and nerve growth occurred only beneath areas with epidermal cover as compared with the surrounding granulation tissue. Initially, the immunoreactivity to von Willebrand factor was high, but in areas with epidermal cover it assumed a more orderly pattern with fewer blood vessels. Innervation was first detected by S-100 immunoreactivity seen at 1 to 2 weeks, closely followed by that to protein gene product 9.5 and much later to calcitonin gene-related peptide. C-flanking peptide of neuropeptide Y and vasoactive intestinal peptide immunoreactivity were detected in the wound depth surrounding large blood vessels at 4 to 6 weeks. In control wounds that had been either grafted with de-epidermalized dermis alone (n = 10) or allowed to granulate (n = 10), persistently there was high immunoreactivity to von Willebrand factor but minimal immunoreactivity to the neural markers. In conclusion, kerato-dermal grafts become innervated, and beneath their surface there is also vascular organization to resemble normal skin. Keratinocytes themselves may influence angiogenesis and innervation, as both processes failed to occur beneath granulating areas.     

The protein translocation apparatus contributes to determining the topology of an integral membrane protein in Escherichia coli

The assembly of integral membrane proteins is determined by features of these proteins and the protein translocation apparatus. We used alkaline phosphatase fusions to the membrane protein MalF to investigate the role of the protein translocation machinery in the arrangement of proteins in the cytoplasmic membrane of Escherichia coli. In particular, we studied the effects of prlA mutations on membrane protein topology. These mutations lie in the secY gene, which encodes a core component of the protein translocation apparatus. We find that the topology of some of the fusion proteins is changed and, in one case, is completely inverted in prlA mutants. We discuss the mechanism of prlA-mediated export and the role of the protein translocation apparatus in contributing to membrane protein topology.     

Biogenesis of the purple membrane of Halobacterium halobium

In 168 eyes with preretinal membranes studied histopathologically, five major distinct types of membranes were categorized: 61 glial, 28 fibrous, 22 cortical vitreous, 16 retinal pigment epithelium, 15 fibroinflammatory, 24 combinations, and one endothelial and melanomatous membrane each. The overall prevalence of preretinal membranes was 5.5% in 2,000 cases studied. Selected cases examined by electron microscopy were supportive of the origin of the cells in the preretinal membranes in the glial, fibrous, and retinal pigment epithelial types. Glial preretinal membranes occurred as the result of defects in the internal limiting membrane, such as retinal pits or holes, and from the optic nerve head in association with posterior vitreous detachment. Fibrous preretinal membranes were associated with proliferative retinopathy and long-standing retinal detachment. Studies of the cellular origin of these membranes were inconclusive. The cortical vitreous type of preretinal membrane was hypocellular and occurred in the absence of associated ocular abnormalities. Pigment epithelial preretinal membranes occurred in eyes with rhegmatogenous retinal detachment.     

The immunochemical approach to the characterization of membrane proteins. Human erythrocyte membrane proteins analysed as a model system

1. Crossed immunoelectrophoresis was used for extensive characterization of individual proteins of human erythrocyte membranes solubilized in non-ionic detergent. 2. The precipitates were assigned to extrinsic or intrinsic proteins. 3. Four glycoproteins were identified by their lectin binding behaviour, whilst five proteins were affected by neuraminidase, indicating them to be sialoglycoproteins. 4. Enzymatic activity is retained in the solubilized system and the presence of acetylcholinesterase and an ATPase was demonstrated. The formation of phosphorylated membrane proteins on incubation with [32P]ATP was demonstrated by autoradiography on the immunoelectrophoresis plates. 5. Five proteins located on the outer cell surface were identified by antibody binding to intact cells. These same proteins were degraded by proteolytic enzymes in intact cells but only three of them were labelled by lactoperoxidase-catalysed 125I-iodination. 6. Analysis of erythrocyte membrane proteins using quantitive immunoelectrophoresis yields results concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis.     

Electrophoretic characterization of proteins in the plasma membrane of porcine spermatozoa

A study of the steady-state kinetics of fumarase over an extended concentration range, using novel methods of analysis, reveals an initial-rate equation of at least fourth degree for malate as substrate at pH 7.0, with no kinetically significant dead-end complex formation even up to concentrations of 100 mM. In the absence of demonstrable enzyme-aggregation phenomena, this is interpreted as indicating co-operative effects overlooked previously, although a mixture of isoenzymes, each individually of high degree and giving a complex curve, may be a contributing factor.     

The potential value of integral membrane proteins in the vaccination of lambs against Haemonchus contortus

Cognitive-behavioral therapy (CBT) is applicable to all eating disorders but has been most intensively studied in the treatment of bulimia nervosa (BN). CBT is designed to alter abnormal attitudes about body shape and weight, replace dysfunctional dieting with normal eating habits, and develop coping skills for resisting binge eating and purging. CBT is effective in reducing all core features of BN and shows good maintenance of therapeutic improvement. Although superior to therapy with antidepressant drugs, CBT has not been shown to be consistently superior to alternative psychological treatments. Different hypotheses about CBT's mechanisms of action are discussed.     

Treatment with crystalline ultra-pure urea reduces the aggregation of integral membrane proteins without inhibiting N-terminal sequencing

We have demonstrated that N-terminal sequencing can be performed successfully despite boiling protein samples in the presence of urea under precise conditions, before loading them onto SDS-PAGE and transfer to polyvinylidene difluoride membrane. Using myoglobin as a test protein, we found that its ability to undergo N-terminal sequencing was not affected by the presence of urea provided "ultra-pure" urea was used. Consistent with this result, we verified that urea did not carbamylate myoglobin since its molecular mass was measured by mass spectrometry after electroelution of the protein band from the gel. These observations are useful for the study of integral membrane proteins, in particular to study their topology from proteolysis experiments, since heating in the presence of urea before SDS-PAGE reduces membrane protein aggregation [Soulié, S., Mo/ller, J.V., Falson, P., and le Maire, M. (1996) Anal. Biochem. 236, 363-364]. We show that the sequencing yield of a hydrophobic peptide from reticulum Ca2+-ATPase was more than doubled in the presence of urea in accord with the quantification of the Coomassie Blue staining of the gel and of the amount present on the polyvinylidene difluoride membrane. For three peptides of the gastric H+K+-ATPase, the sequencing yield after urea treatment increased almost threefold.     

Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration.     

Characterization of the human SDHC gene encoding of the integral membrane proteins of succinate-quinone oxidoreductase in mitochondria

The neurological manifestations of multiple sclerosis (MS) have been considered to result from demyelination of axons with relative preservation of axonal integrity. This concept has been challenged recently by a landmark pathological study, published in the New England Journal of Medicine, which has demonstrated that axonal degeneration is also present. The authors of the study hypothesized that axonal degeneration is the pathological correlate of the irreversible neurological impairment in this disease. However, this hypothesis cannot be reconciled with the clinical results obtained with transcranial applications of AC pulsed electromagnetic fields (EMFs) of picotesla flux density which have shown rapid and sustained improvement of symptoms including normalization of evoked potential responses in patients with chronic progressive or secondary progressive MS without demyelinated areas first undergoing remyelination or transected axons undergoing regeneration. Biochemical studies have shown that MS patients are serotonergically depleted with the extent of cerebral depletion correlating with the degree of motor disability and a chronic progressive course. It is believed that progressive serotonergic neuronal atrophy with synaptic inactivation, not axonal degeneration, are the hallmarks of the disease and that administration of AC pulsed magnetic fields improves symptoms of MS partly through reactivation of serotonergic neurons and amplification of synaptic serotonergic transmission.     

Identification and characterization of golgin-84, a novel Golgi integral membrane protein with a cytoplasmic coiled-coil domain

The cytoplasmic face of the Golgi contains a variety of proteins with coiled-coil domains. We identified one such protein in a yeast two-hybrid screen, using as bait the peripheral Golgi phosphatidylinositol(4,5)P2 5-phosphatase OCRL1 that is implicated in a human disease, the oculocerebrorenal syndrome. The approximately 2.8-kilobase mRNA is ubiquitously expressed and abundant in testis; it encodes a 731-amino acid protein with a predicted mass of 83 kDa. Antibodies against the sequence detect a novel approximately 84-kDa Golgi protein we termed golgin-84. Golgin-84 is an integral membrane protein with a single transmembrane domain close to its C terminus. In vitro, the protein inserts post-translationally into microsomal membranes with an N-cytoplasmic and C-lumen orientation. Cross-linking indicates that golgin-84 forms dimers, consistent with the prediction of an approximately 400-residue dimerizing coiled-coil domain in its N terminus. The dimerization potential is supported by a data base search that showed that the N-terminal 497 residues of golgin-84 contain a coiled-coil domain that when fused to the RET tyrosine kinase domain had the ability to activate it, forming the RET-II oncogene. Data base searching also indicates golgin-84 is similar in structure and sequence to giantin, a membrane protein that tethers coatamer complex I vesicles to the Golgi.     

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.     

Assessment of the aggregation state of integral membrane proteins in reconstituted phospholipid vesicles using small angle neutron scattering

The assessment of the physical size of integral membrane protein complexes has generally been limited to samples solubilized in non-ionic detergent, a process which may introduce artifacts of unknown scope and severity. A system has been developed that allows observation of the small angle scattering profile of an integral membrane protein while incorporated in small unilamellar phospholipid vesicles. Contrast matching of isotopically substituted phospholipid eliminates the contribution of the bilayer to the observed scattering, resulting in a profile dependent only on the structure of the individual membrane protein complexes and their spatial arrangement in the vesicle. After appropriate compensation for their spatial arrangement, information about the molecular mass and radius of gyration of the individual complexes can be obtained. The validity of the approach has been established using monomeric bacteriorhodopsin as a model system.     

Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3-1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61-2 allele. This is accompanied by the stabilization of the Sec61-2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61-2 strain at the permissive temperature of 25 degrees C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61-2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.     

MAL, a novel integral membrane protein of human T lymphocytes, associates with glycosylphosphatidylinositol-anchored proteins and Src-like tyrosine kinases

A large fraction of glycosylphosphatidylinositol (GPI)-anchored proteins and Src-like kinases are confined to glycolipid-enriched membrane (GEM) microdomains. The particular membrane topology of GPI-anchored proteins has led to the postulation of the existence of integral membrane proteins linking extracellular stimuli with cytosolic machinery for endocytosis and signaling. The human MAL cDNA was identified during a search for novel genes differentially expressed during T cell development, and encodes a multispanning membrane protein displaying lipid-like properties. To address the biochemical characterization of endogenous MAL and to analyze its possible association with other proteins, we have generated a monoclonal antibody (mAb) specific to the MAL molecule. Using this mAb, we have identified MAL in GEM microdomains of both the HPB-ALL T cell line and human peripheral blood lymphocytes. Co-immunoprecipitation experiments with antibodies to the MAL molecule or to the GPI-anchored CD59 antigen indicated specific association of MAL with GPI-anchored proteins and Src-like tyrosine kinases. In addition, both MAL and the Src-like kinase Lck were identified in GEM obtained from an endosomal-enriched membrane fraction. These features of MAL closely match some of the properties expected for the hypothetical integral membrane linker proteins acting in specialized GEM-mediated functions.     

Semlike Forest virus membrane proteins. Preparation and characterization of spike complexes soluble in detergent-free medium

After Triton X-100 delipidation and subsequent Triton X-100 removal in a sucrose gradient the membrane protein spikes of Semliki Forest virus remained soluble in aqueous buffers. It was shown they were present as octameric complexes with a molecular weight of 95-10(4) and that they contain less than 4% lipid and detergent by weight. In electron microscopy after negative staining they appeared as "rosette"-shaped particles. Part of the protein could also be found associated in ordered paracrystalline arrays.