Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.     

Protein transport across the endoplasmic reticulum membrane

A decisive step in the biosynthesis of many eukaryotic proteins is their partial or complete translocation across the endoplasmic reticulum membrane. A similar process occurs in prokaryotes, except that proteins are transported across or are integrated into the plasma membrane. In both cases, translocation occurs through a protein-conducting channel that is formed from a conserved, heterotrimeric membrane protein complex, the Sec61 or SecY complex. Structural and biochemical data suggest mechanisms that enable the channel to function with different partners, to open across the membrane and to release laterally hydrophobic segments of membrane proteins into lipid.     

Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria

Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.     

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.     

Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.     

Identification of novel families of membrane proteins from the model plant Arabidopsis thaliana

MOTIVATION: The completion of the Arabidopsis genome offers the first opportunity to analyze all of the membrane protein sequences of a plant. The majority of integral membrane proteins including transporters, channels, and pumps contain hydrophobic alpha-helices and can be selected based on TransMembrane Spanning (TMS) domain prediction. By clustering the predicted membrane proteins based on sequence, it is possible to sort the membrane proteins into families of known function, based on experimental evidence or homology, or unknown function. This provides a way to identify target sequences for future functional analysis. RESULTS: An automated approach was used to select potential membrane protein sequences from the set of all predicted proteins and cluster the sequences into related families. The recently completed sequence of Arabidopsis thaliana, a model plant, was analyzed. Of the 25,470 predicted protein sequences 4589 (18%) were identified as containing two or more membrane spanning domains. The membrane protein sequences clustered into 628 distinct families containing 3208 sequences. Of these, 211 families (1764 sequences) either contained proteins of known function or showed homology to proteins of known function in other species. However, 417 families (1444 sequences) contained only sequences with no known function and no homology to proteins of known function. In addition, 1381 sequences did not cluster with any family and no function could be assigned to 1337 of these.     

Novel mechanisms for the targeting of proteins into and across chloroplast membranes

The assembly of the photosynthetic apparatus requires the translocation of numerous proteins from the cytosol, initially into the stroma and thereafter into or across the thylakoid membrane. Recent studies have shown that proteins are transported into this membrane by a variety of mechanisms, some of which are derived from a cyanobacterial-type ancestor, whereas others have evolved in response to the more complex transport pathway used by cytosolically synthesized chloroplast proteins. It is now apparent that some of the targeting pathways are used exclusively by hydrophobic thylakoid membrane proteins; here we review recent progress in our understanding of the biogenesis of this important class of protein.     

Differential sorting of constitutively co-secreted proteins in the ovarian follicle cells of Drosophila

Conventional and freeze-fracture electron microscopy, immuno-electron microscopy of ovarian cryosections and confocal immunofluorescence were used to analyze the ovarian distribution of the major protein classes being secreted by the follicle cells during the vitellogenic and choriogenic stages of Drosophila oogenesis. Our results clearly demonstrated that at vitellogenic stages the follicle cells co-secrete constitutively vitelline membrane and yolk proteins that are either sorted into distinct secretory vesicles or they are segregated in different parts of bipartite vesicles by differential condensation. Following their exocytosis only the vitelline membrane proteins are incorporated into the forming vitelline membrane. The yolk proteins (along with their hemolymph circulating counterparts) diffuse through gaps amongst the incomplete vitelline membrane and are internalized through endocytosis by the oocyte where they are finally stored into modified lysosomes referred to as alpha-yolk granules. The unexpected immunolocalization of vitelline membrane antigens in the associated body of the alpha-yolk granules may indicate that this structure is a transient repository for the proteins being internalized into the oocyte along with the yolk proteins. In the early choriogenic follicle cells the vitelline membrane and early chorion proteins were found to be co-secreted and to be evenly intermixed into the same secretory vesicles. These findings illuminate new details concerning the follicle cells secretory and oocyte endocytic pathways and provide for the first time evidence for condensation-mediated sorting of constitutively secreted proteins in Drosophila.     

MemO: a consensus approach to the annotation of a protein's membrane organization

Membrane organization describes the relationship of proteins to the membrane, that is, whether the protein crosses the membrane or is integral to the membrane and its orientation with respect to the membrane. Membrane organization is determined primarily by the presence of two features which target proteins to the secretory pathway: the endoplasmic reticulum signal peptide and the ?-helical transmembrane domain. In order to generate membrane organization annotation of high quality, confidence and throughput, the Membrane Organization (MemO) pipeline was developed, incorporating consensus feature prediction modules with integration and annotation rules derived from biological observations. The pipeline classifies proteins into six categories based on the presence or absence of predicted features: Soluble, intracellular proteins; Soluble, secreted proteins; Type I membrane proteins; Type II membrane proteins; Multi-span membrane proteins and Glycosylphosphatidylinositol anchored membrane proteins. The MemO pipeline represents an integrated strategy for the application of state-of-the-art bioinformatics tools to the annotation of protein membrane organization, a property which adds biological context to the large quantities of protein sequence information available.     

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle

Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.     

Reconstitution of the partially purified renal phosphate (Pi) transporter.

Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.     

Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria

Many metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.     

Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix

The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria. Little is known about the intramitochondrial sorting of these proteins. We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space. Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane. The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane. The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria. A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.     

The structure of the Sec complex and the problem of protein translocation

Proteins synthesized in the cytosol either remain there or are localized to a specific membrane and subsequently translocated to another cellular compartment. These extracytosolic proteins have to cross, or be inserted into, a phospholipid bilayer-a process governed by membrane-bound protein transporters designed to recognize and receive appropriate polypeptides and thread them through the membrane. One such translocation complex, SecY/Sec61, is found in every cell, in either the plasma membrane of bacteria and archaea or the endoplasmic reticulum membrane of eukaryotes. Recent structural findings, combined with previous genetic and biochemical studies, have helped to describe how the passage of proteins through the membrane might occur, but several points of uncertainty remain.     

Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion

Current models for membrane fusion in diverse biological processes are focused on the local action of fusion proteins present in the contact zone where the proteins anchored in one membrane might interact directly with the other membrane. Are the fusion proteins outside of the contact zone just bystanders? Here we assess the role of these "outsider" proteins in influenza virus hemagglutinin-mediated fusion between red blood cells and either hemagglutinin-expressing cells or viral particles. To selectively inhibit or enhance the actions of hemagglutinin outsiders, the antibodies that bind to hemagglutinin and proteases that cleave it were conjugated to polystyrene microspheres too large to enter the contact zone. We also involved hemagglutinin outsiders into interactions with additional red blood cells. We find the hemagglutinin outsiders to be necessary and sufficient for fusion. Interfering with the activity of the hemagglutinin outsiders inhibited fusion. Selective conversion of hemagglutinin outsiders alone into fusion-competent conformation was sufficient to achieve fusion. The discovered functional role of fusion proteins located outside of the contact zone suggests a tempting analogy to mechanisms by which proteins mediate membrane fission from outside of the fission site.     

Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530). Progress has been made in the elucidation of how these proteins are targeted to the organelle. In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly. However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited. The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles. Furthermore, the origin of the peroxisomal membrane is still enigmatic. Recent observations suggest the existence of two classes of peroxisomal membrane proteins. Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli.

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precurser pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [35S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s. The [35S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30--120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation. Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Lipid rafts and the regulation of exocytosis

Exocytosis is the process whereby intracellular fluid-filled vesicles fuse with the plasma membrane, incorporating vesicle proteins and lipids into the plasma membrane and releasing vesicle contents into the extracellular milieu. Exocytosis can occur constitutively or can be tightly regulated, for example, neurotransmitter release from nerve endings. The last two decades have witnessed the identification of a vast array of proteins and protein complexes essential for exocytosis. SNARE proteins fill the spotlight as probable mediators of membrane fusion, whereas proteins such as munc18/nsec1, NSF and SNAPs function as essential SNARE regulators. A central question that remains unanswered is how exocytic proteins and protein complexes are spatially regulated. Recent studies suggest that lipid rafts, cholesterol and sphingolipid-rich microdomains, enriched in the plasma membrane, play an essential role in regulated exocytosis pathways. The association of SNAREs with lipid rafts acts to concentrate these proteins at defined sites of the plasma membrane. Furthermore, cholesterol depletion inhibits regulated exocytosis, suggesting that lipid raft domains play a key role in the regulation of exocytosis. This review examines the role of lipid rafts in regulated exocytosis, from a passive role as spatial coordinator of exocytic proteins to a direct role in the membrane fusion reaction.     

Making the Most of Fusion Tags Technology in Structural Characterization of Membrane Proteins

Membrane proteins can be investigated at various structural levels, including the topological structure, the high-resolution three-dimensional structure, and the organization and assembly of membrane protein complexes. Gene fusion technology makes it possible to insert a polynucleotide encoding a protein or polypeptide tag into the gene encoding a membrane protein of interest. Resultant recombinant proteins may possess the functions of the original membrane proteins, together with the biochemical properties of the imported fusion tag, greatly enhancing functional and structural studies of membrane proteins. In this article, the latest literature is reviewed in relation to types, applications, strategies, and approaches to fusion tag technology for structural investigations of membrane proteins.     

Alteration of the Escherichia coli membrane by addition of bacteriophage T4 protein synthesized after infection.

Many T4-induced proteins were found associated with the Escherichia coli membrane during infection. Some of these were apparently ionically bound, but many could be identified as integral parts of the inner and outer bacterial membranes by their selective solubilities in guanidine or Sarkosyl. The synthesis and insertion of these proteins into the bacterial membrane were temporally controlled and, once in the membrane, these proteins were stably integrated. Host membrane protein synthesis continued after infection of non-UV-irradiated cells, but was not present, if the cells were irradiated. There were no major redistribution or loss of bacterial proteins from E. coli membranes as a consequence of T4 infection.