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Human growth hormone (hGH) is a four‐helix bundle protein of considerable pharmacological interest. Recombinant hGH is produced in bacteria, yet little is known about its folding during expression in Escherichia coli. We have studied the cotranslational folding of hGH using force profile analysis (FPA), both during in vitro translation in the absence and presence of the chaperone trigger factor (TF), and when expressed in E. coli. We find that the main folding transition starts before hGH is completely released from the ribosome, and that it can interact with TF and possibly other chaperones. [ABSTRACT FROM AUTHOR]

During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~ 45 residues away from the peptidyl transferase centre. However, we recently uncovered instances where the first contact is delayed by up to ~ 10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the Escherichia coli inner membrane protein BtuC: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion. [ABSTRACT FROM AUTHOR]

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm. [ABSTRACT FROM AUTHOR]

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite‐derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well‐established experimental system that uses N‐linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt‐Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen. [ABSTRACT FROM AUTHOR]

The TIM23 complex mediates membrane insertion of presequence‐containing mitochondrial proteins via a stop‐transfer mechanism. Stop‐transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop‐transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23‐mediated stop‐transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2‐deletion or ‐overexpression yeast strains. We found that upon deletion of Mgr2, the threshold hydrophobicity for membrane insertion, as well as the requirement for matrix‐facing positive charges, is reduced. These results imply that the Mgr2‐mediated gatekeeper function is important for controlling membrane sorting of marginal stop‐transfer signals. [ABSTRACT FROM AUTHOR]

Recent work has demonstrated that cotranslational folding of proteins or protein domains in, or in the immediate vicinity of, the ribosome exit tunnel generates a pulling force on the nascent polypeptide chain that can be detected using a so-called translational arrest peptide ( AP) engineered into the nascent chain as a force sensor. Here, we show that AP-based force measurements combined with systematic Ala and Trp scans of a zinc-finger domain that folds in the exit tunnel can be used to identify the residues that are critical for intraribosomal folding. Our results suggest a general approach to characterize the folded state(s) that may form as a protein domain moves progressively down the ribosome exit tunnel. [ABSTRACT FROM AUTHOR]

The divisome is the macromolecular complex that carries out cell division in Escherichia coli. Every generation it must be assembled, and then disassembled so that the sequestered proteins can be recycled. Whilst the assembly process has been well studied, virtually nothing is known about the disassembly process. In this study, we have used super-resolution SIM imaging to monitor pairs of fluorescently tagged divisome proteins as they depart from the division septum. These simple binary comparisons indicated that disassembly occurs in a coordinated process that consists of at least five steps: [FtsZ, ZapA] ⇒ [ZipA, FtsA] ⇒ [FtsL, FtsQ] ⇒ [FtsI, FtsN] ⇒ [FtsN]. This sequence of events is remarkably similar to the assembly process, indicating that disassembly follows a first-in, first-out principle. A secondary observation from these binary comparisons was that FtsZ and FtsN formed division rings that were spatially separated throughout the division process. Thus the data indicate that the divisome structure can be visualized as two concentric rings; a proto-ring containing FtsZ and an FtsN-ring. [ABSTRACT FROM AUTHOR]

Cotranslational folding of small protein domains within the ribosome exit tunnel may be an important cellular strategy to avoid protein misfolding. However, the pathway of cotranslational folding has so far been described only for a few proteins, and therefore, it is unclear whether folding in the ribosome exit tunnel is a common feature for small protein domains. Here, we have analyzed nine small protein domains and determined at which point during translation their folding generates sufficient force on the nascent chain to release translational arrest by the SecM arrest peptide, both in vitro and in live E. coli cells. We find that all nine protein domains initiate folding while still located well within the ribosome exit tunnel. [ABSTRACT FROM AUTHOR]

Highlights: [•] We measured co-translational pulling forces on Nin-facing transmembrane segments. [•] The pulling forces are weaker than for Nout-orientated transmembrane segments. [•] The biphasic shape of force profiles from Nin- and Nout-segments is preserved. [ABSTRACT FROM AUTHOR]

In most bacteria cell division is mediated by a protein super-complex called the divisome that co-ordinates the constriction and scission of the cell envelope. FtsZ is the first of the divisome proteins to accumulate at the division site and is widely thought to function as a force generator that constricts the cell envelope. In this study we have used a combination of confocal fluorescence microscopy and fluorescence recovery after photobleaching ( FRAP) to determine if divisome proteins are present at the septum at the time of cytoplasmic compartmentalization in E scherichia coli. Our data suggest that many are, but that FtsZ and ZapA disassemble before the cytoplasm is sealed by constriction of the inner membrane. This observation implies that FtsZ cannot be a force generator during the final stage(s) of envelope constriction in E . coli. [ABSTRACT FROM AUTHOR]

Highlights: [•] C-terminal extensions to Sdh3 and Shh3 result in SRP-dependent ER mis-targeting. [•] Mitochondrial membrane proteins evade SRP recognition by having short C-terminal tails after strongly hydrophobic segments. [•] Eluding SRP recognition may be one way that hydrophobic membrane proteins ensure proper targeting to mitochondria. [ABSTRACT FROM AUTHOR]

Highlights: [•] Membrane proteins are extremely challenging to produce. [•] We attempted to improve expression of two difficult to express coding sequences. [•] Multi-parameter sequence optimization of codons and other variables failed. [•] Substitution of synonymous codons adjacent to the AUG start worked efficiently. [•] Coding sequences can be re-wired for higher expression by selective engineering. [Copyright &y& Elsevier]

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structure-function relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli. [ABSTRACT FROM AUTHOR]

Abstract: Mgm1p is a nuclearly encoded GTPase important for mitochondrial fusion. Long and short isoforms of the protein are generated in a unique “alternative topogenesis” process in which the most N-terminal of two hydrophobic segments in the protein is inserted into the inner mitochondrial membrane in about half of the molecules and translocated across the inner membrane in the other half. In the latter population, the second hydrophobic segment is cleaved by the inner membrane protease Pcp1p, generating the short isoform. Here, we show that charged residues in the regions flanking the first segment critically affect the ratio between the two isoforms, providing new insight into the importance of charged residues in the insertion of proteins into the mitochondrial inner membrane. [Copyright &y& Elsevier]

We present a fluorescence recovery after photobleaching-based method for monitoring the progression of septal Z-ring contraction in dividing Escherichia coli cells. In a large number of cells undergoing division, we irreversibly bleached cytosolically expressed Enhanced Green Fluorescent Protein on one side of the septal invagination and followed the fluorescence relaxation on both sides of the septum. Since the relaxation time depends on the cross-sectional area of the septum, it can be used to determine the septal radius r. Assuming that the fraction of the observed cells with r-values in a given interval reflects the duration of that interval in the division process we could derive an approximate time-course for the contraction event, as a population average. By applying the method repeatedly on individual cells, the contraction process was also followed in real time. On a population average level, our data are best described by a linear contraction process in time. However, on the single cell level the contraction processes display a complex behaviour, with varying levels of activity. The proposed approach provides a simple yet versatile method for studying Z-ring contraction in vivo, and will help to elucidate its underlying mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: By analogy to mammals, odorant receptors (ORs) in insects, such as Drosophila melanogaster, have long been thought to belong to the G-protein coupled receptor (GPCR) superfamily. However, recent work has cast doubt on this assumption and has tentatively suggested an inverted topology compared to the canonical N out − C in 7 transmembrane (TM) GPCR topology, at least for some Drosophila ORs. Here, we report a detailed topology mapping of the Drosophila OR83b receptor using engineered glycosylation sites as topology markers. Our results are inconsistent with a classical GPCR topology and show that OR83b has an intracellular N-terminus, an extracellular C-terminus, and 7TM helices. [Copyright &y& Elsevier]

Abstract: Alzheimer’s disease is characterized by the deposition of amyloid β-peptide (Aβ) plaques in the brain. Full-length amyloid-β precursor protein (APP) is processed by α- and β-secretases to yield soluble APP derivatives and membrane-bound C-terminal fragments, which are further processed by γ-secretase to a non-amyloidogenic 3kDa product or to Aβ fragments. As different Aβ fragments contain different parts of the APP transmembrane helix, one may speculate that they are retained more or less efficiently in the membrane. Here, we use the translocon-mediated insertion of different APP-derived polypeptide segments into the endoplasmic reticulum membrane to assess the propensities for membrane retention of Aβ fragments. Our results show a strong correlation between the length of an Aβ-derived segment and its ability to integrate into the microsomal membrane. [Copyright &y& Elsevier]

Abstract: The Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene encodes an integral membrane protein, called seipin, of unknown function localized to the endoplasmic reticulum of eukaryotic cells. Seipin is associated with the heterogeneous genetic disease BSCL2, and mutations in an N-glycosylation motif links the protein to two other disorders, autosomal-dominant distal hereditary motor neuropathy type V and Silver syndrome. Here, we report a topological study of seipin using an in vitro topology mapping assay. Our results suggest that the predominant form of seipin is 462 residues long and has an Ncyt–Ccyt orientation with a long luminal loop between the two transmembrane helices. [Copyright &y& Elsevier]

Many new Escherichia coli outer membrane proteins have recently been identified by proteomics techniques. However, poorly expressed proteins and proteins expressed only under certain conditions may escape detection when wild-type cells are grown under standard conditions. Here, we have taken a complementary approach where candidate outer membrane proteins have been identified by bioinformatics prediction, cloned and overexpressed, and finally localized by cell fractionation experiments. Out of eight predicted outer membrane proteins, we have confirmed the outer membrane localization for five-YftM, YaiO, YfaZ, CsgF, and YliI-and also provide preliminary data indicating that a sixth-YfaL-may be an outer membrane autotransporter. [ABSTRACT FROM AUTHOR]

We have performed a comparative analysis of amino acid distributions in predicted integral membrane proteins from a total of 107 genomes. A procedure for identification of membrane spanning helices was optimized on a homology-reduced data set of 170 multi-spanning membrane proteins with experimentally determined topologies. The optimized method was then used for extraction of highly reliable partial topologies from all predicted membrane proteins in each genome, and the average biases in amino acid distributions between loops on opposite sides of the membrane were calculated. The results strongly support the notion that a biased distribution of Lys and Arg residues between cytoplasmic and extra-cytoplasmic segments (the positive-inside rule) is present in most if not all organisms. Proteins 2005. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Topology predictions for integral membrane proteins can be substantially improved if parts of the protein can be constrained to a given in/out location relative to the membrane using experimental data or other information. Here, we have identified a set of 367 domains in the SMART database that, when found in soluble proteins, have compartment-specific localization of a kind relevant for membrane protein topology prediction. Using these domains as prediction constraints, we are able to provide high-quality topology models for 11% of the membrane proteins extracted from 38 eukaryotic genomes. Two-thirds of these proteins are single spanning, a group of proteins for which current topology prediction methods perform particularly poorly. [ABSTRACT FROM AUTHOR]

To better define the mechanism of membrane protein insertion into the membrane of the endoplasmic reticulum, we measured the kinetics of translocation across microsomal membranes of the N-terminal lumenal tail and the lumenal domain following the second transmembrane segment (TM2) in the multispanning mouse protein Cig30. In the wild-type protein, the N-terminal tail translocates across the membrane before the downstream lumenal domain. Addition of positively charged residues to the N-terminal tail dramatically slows down its translocation and allows the downstream lumenal domain to translocate at the same time as or even before the N-tail. When TM2 is deleted, or when the loop between TM1 and TM2 is lengthened, addition of positively charged residues to the N-terminal tail causes TM1 to adopt an orientation with its N-terminal end in the cytoplasm. We suggest that the topology of the TM1-TM2 region of Cig30 depends on a competition between TM1 and TM2 such that the transmembrane segment that inserts first into the ER membrane determines the final topology. [ABSTRACT FROM AUTHOR]

Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies. [ABSTRACT FROM AUTHOR]

We report the development of LumenP, a new neural network-based predictor for the identification of proteins targeted to the thylakoid lumen of plant chloroplasts and prediction of their cleavage sites. When used together with the previously developed TargetP predictor, LumenP reaches a significantly better performance than what has been recorded for previous attempts at predicting thylakoid lumen location, mostly due to a lower false positive rate. The combination of TargetP and LumenP predicts around 1.5%-3% of all proteins encoded in the genomes of Arabidopsis thaliana and Oryza sativa to be located in the lumen of the thylakoid. [ABSTRACT FROM AUTHOR]

A method to predict lipoprotein signal peptides in Gram-negative Eubacteria, LipoP, has been developed. The hidden Markov model (HMM) was able to distinguish between lipoproteins (SPaseII-cleaved proteins), SPaseI-cleaved proteins, cytoplasmic proteins, and transmembrane proteins. This predictor was able to predict 96.8% of the lipoproteins correctly with only 0.3% false positives in a set of SPaseI-cleaved, cytoplasmic, and transmembrane proteins. The results obtained were significantly better than those of previously developed methods. Even though Gram-positive lipoprotein signal peptides differ from Gram-negatives, the HMM was able to identify 92.9% of the lipoproteins included in a Gram-positive test set. A genome search was carried out for 12 Gram-negative genomes and one Gram-positive genome. The results for Escherichia coli K12 were compared with new experimental data, and the predictions by the HMM agree well with the experimentally verified lipoproteins. A neural network-based predictor was developed for comparison, and it gave very similar results. LipoP is available as a Web server at . [ABSTRACT FROM AUTHOR]

We have developed a method to reliably identify partial membrane protein topologies using the consensus of five topology prediction methods. When evaluated on a test set of experimentally characterized proteins, we find that approximately 90% of the partial consensus topologies are correctly predicted in membrane proteins from prokaryotic as well as eukaryotic organisms. Whole-genome analysis reveals that a reliable partial consensus topology can be predicted for ∼70% of all membrane proteins in a typical bacterial genome and for ∼55% of all membrane proteins in a typical eukaryotic genome. The average fraction of sequence length covered by a partial consensus topology is 44% for the prokaryotic proteins and 17% for the eukaryotic proteins in our test set, and similar numbers are found when the algorithm is applied to whole genomes. Reliably predicted partial topologies may simplify experimental determinations of membrane protein topology. [ABSTRACT FROM AUTHOR]

A total of 20%-25% of the proteins in a typical genome are helical membrane proteins. The transmembrane regions of these proteins have markedly different properties when compared with globular proteins. This presents a problem when homology search algorithms optimized for globular proteins are applied to membrane proteins. Here we present modifications of the standard Smith-Waterman and profile search algorithms that significantly improve the detection of related membrane proteins. The improvement is based on the inclusion of information about predicted transmembrane segments in the alignment algorithm. This is done by simply increasing the alignment score if two residues predicted to belong to transmembrane segments are aligned with each other. Benchmarking over a test set of G-protein-coupled receptor sequences shows that the number of false positives is significantly reduced in this way, both when closely related and distantly related proteins are searched for. [ABSTRACT FROM AUTHOR]

Describes the N-translocation of a polytopic membrane protein. Comparison of N-tail translocation in the 4R-mutants versus wild-type cig30; Ability of transmembrane1 (TM1) to induce translocation of the wild-type N-tail; Inability of TM1 to induce translocation in 4R mutants; Required presence of TM1 and TM2 for translocation of the N-tail in the 4R-9 mutant.

We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level is well above that of the publicly available chloroplast localization predictor PSORT. Cleavage sites are predicted using a scoring matrix derived by an automatic motif-finding algorithm. Approximately 60% of the known cleavage sites in our sequence collection were predicted to within ±2 residues from the cleavage sites given in SWISS-PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS-PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome-wide sequence data. The ChloroP predictor is available as a web-server at http://www.cbs.dtu.dk/services/ChloroP/. [ABSTRACT FROM PUBLISHER]

Three models for mRNA translation are discussed in the light of available experimental data. It is concluded that the elongation rates vary along a messenger, possibly as a result of a coupling between ribosome movement and mRNA secondary structure. Some promising areas of further experimentation are indicated. [ABSTRACT FROM AUTHOR]

Some recent trends in the development of theoretical methods for DNA and protein sequence analysis are reviewed, with particular emphasis on the design of new databases, motif searches, sequence alignment algorithms and applications of neural networks. [ABSTRACT FROM AUTHOR]

Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character. No highly conserved 'homology blocks' are found in either class of topogenic sequence. Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties. Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases. In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic β-strand. Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an aminoterminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides. [ABSTRACT FROM AUTHOR]

A new middle repetitive sequence is described in the mouse genome. It has been revealed with a recombinant clone isolated from a Mus musculus BamHI gene library constructed in pBR322 and containing an insertion of 1.73 kb. When digests of genomic DNA were subjected to Southern blot hybridization, using the 1.73-kb insert as probe, we obtained a light smear and discrete bands, indicating a dispersion in the mouse genome of this sequence. This 1.73-kb sequence seems to be a part of a greater repetitive sequence at least 6 kb in length. The sizes of the bands hybridizing with the 1.73-kb insert are similar when compared between different laboratory strains but differ remarkably between the two species M. musculus and Mus caroli. We have shown also a great variation in the copy number of the sequence studied between these two species. When rat DNA is probed with the 1.73-kb insert, no hybridization is observed. Subcloning of the 1.73-kb sequence in three fragments has pointed out that the reiteration was not homogeneous along the 1.73-kb sequence. The 1.73-kb clone was sequenced and compared with other interspersed repetitive sequences, previously described in the rodent genome, and no homology was found. Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information. [ABSTRACT FROM AUTHOR]

According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side-chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made. [ABSTRACT FROM AUTHOR]

The overall amino acid composition of membrane-penetrating segments of trans-membrane proteins can be well reproduced by computer simulation of `evolution' of hydrophobic protein segments, with selection operating only on the hydrophobicity of the sequence. This substantiates the idea that the specific amino acid sequence is relatively less important than the preservation of an overall hydrophobic character in such segments, and furnishes an example of macromolecular evolution where the evolutionary process can be quantitatively mimicked on the computer. [ABSTRACT FROM AUTHOR]

A number of signal sequences, prokaryotic as well as eukaryotic, have been analyzed in terms of gross amino acid composition and hydrophobicity. It is shown that the amino acid composition of the hydrophic core can be well reproduced in a computer simulation of signal sequence ‘evolution’ with selection operating on the mean hydrophobicity of the sequence and the non-occurrence of charged residues. The calculated hydrophobicities are interpreted in terms of a model in which the hydrophobic part of the signal sequence partitions directly into the membrane interior, thereby making further translocation of the growing nascent chain possible. [ABSTRACT FROM AUTHOR]

A detailed, computerized procedure for analyzing the translocation process of any protein with known sequence from a physico-chemical point of view is presented and used to gain a better under- standing of the molecular ‘rules’ that govern the final outcome of the process. With the aid of this procedure a number of testable predictions of the orientations of particular membrane-bound proteins can be made. It is also suggested that ovalbumin, the only known secreted protein lacking a cleavable prepiece, initiates translocation in a way that differs from other secreted proteins. [ABSTRACT FROM AUTHOR]

As a start towards a deeper understanding of the transmembrane transport of proteins, the transfer of a nascent chain through the lipophilic core of a membrane is discussed from a physico-chemical point of view. Some simple considerations of the energetics of protein structure, together with experimental data on the transfer process, form the basis for a detailed and quantifiable model, accounting for the extrusion of secreted proteins as well as for the insertion of trans-membrane proteins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have analyzed the amino acid distribution in seven nuclearly encoded and five mitochondrially encoded inner membrane proteins with experimentally well characterized topologies. The mitochondrially encoded proteins conform to the ‘positive inside’ rule, i.e. they have many more positively charged residues in their non-translocated as compared to translocated domains. However, most of the nuclearly encoded proteins do not show such a bias but instead have a surprisingly skewed distribution of Glu residues with an almost ten times higher frequency in the intermembrane space than in the matrix domains. These findings suggest that some, but possibly not all, nuclearly encoded inner membrane proteins may insert into the membrane by a mechanism that does not depend on the distribution of positively charged amino acids. [ABSTRACT FROM AUTHOR]

We have measured the efficiency of stop-transfer function for a set of pseudo-random, 18-residue amino acid segments, both in Escherichia coli and in mammalian microsomes. In general, stop-transfer function correlates well with the mean hydrophobicity of the segment, though exceptions exist. Kinetic studies suggest that polar segments are rapidly translocated through the E. coli inner membrane and that strongly hydrophobic segments become permanently anchored, while sequences with an intermediate mean hydrophobicity become partly trapped in a transmembrane disposition for a considerable time before being released to the periplasm or degraded. [ABSTRACT FROM AUTHOR]

We have analyzed the structure of mitochondrial cytochrome c oxidase in terms of general characteristics thought to be important for describing the architecture of helix bundle membrane proteins. Many aspects of the structure are similar to what has previously been found for the photosynthetic reaction center and bacteriorhodopsin. Our results lead to a considerably more precise general picture of membrane protein architecture than has hitherto been possible to obtain. [ABSTRACT FROM AUTHOR]

The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome. [ABSTRACT FROM AUTHOR]

Many integral membrane proteins use the same translocation machinery for membrane insertion as secretory proteins use to get across the membrane. This requires that transmembrane segments can be discriminated from other parts of the protein during membrane translocation, and further requires that the transmembrane segments can be moved laterally out of the translocation channel into the surrounding lipid. The molecular basis for this remarkable intramembraneous sorting event is a major focus of current studies of membrane protein biogenesis. [ABSTRACT FROM AUTHOR]

N-terminal methionine removal has been analyzed statistically in a large sample of prokaryotic and eukaryotic cytosolic proteins in an attempt to uncover common sequence determinants. We find that the residue next to the initiator Met is the most important determinant of N-terminal processing: Lys, Arg, Leu and (in prokaryotes) Phe and Ile protect the initiator Met from being removed when next to it in the sequence; Ala, Gly, Pro, Ser, Thr and (in eukaryotes) Val in this position cause its removal. Subsequent acetylation is confirmed to be strongly biased towards Ala, Met and Ser residues; when Met is acetylated, Asp is the predominant penultimate residue in eukaryotes. Also, we find major differences in the relative abundance of the various residues next to the initiator Met between prokaryotes and eukaryotes: prokaryotic proteins are much more biased towards Lys as the Met-protecting residue, and towards Ala when Met is to be removed, than eukaryotic ones. Finally, we show that our results can explain a part of the mRNA 'consensus sequence' found around eukaryotic initiator AUG codons. [ABSTRACT FROM AUTHOR]