Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coordinated translocation and membrane integration events. For the human aquaporin water channel AQP1, however, the initial four-segment-spanning topology at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follow the transmembrane (TM) orientation of key internal peptide loops in Xenopus oocyte and cell-free systems. This analysis revealed that AQP1 maturation in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4-6, TM3 underwent a 180-degree rotation in which TM3 C-terminal flanking residues were translocated from their initial cytosolic location into the ER lumen and N-terminal flanking residues underwent retrograde translocation from the ER lumen to the cytosol. These events convert TM3 from a type I to a type II topology and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological reorientation was also associated with maturation from a protease-sensitive conformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cotranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.     

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.     

The membrane insertase YidC

The membrane insertases YidC–Oxa1–Alb3 provide a simple cellular system that catalyzes the transmembrane topology of newly synthesized membrane proteins. The insertases are composed of a single protein with 5 to 6 transmembrane (TM) helices that contact hydrophobic segments of the substrate proteins. Since YidC also cooperates with the Sec translocase it is widely involved in the assembly of many different membrane proteins including proteins that obtain complex membrane topologies. Homologues found in mitochondria (Oxa1) and thylakoids (Alb3) point to a common evolutionary origin and also demonstrate the general importance of this cellular process. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Sec61alpha and TRAM are sequentially adjacent to a nascent viral membrane protein during its ER integration

Co-translational integration of a nascent viral membrane protein into the endoplasmic reticulum membrane takes place via the translocon. We have been studying the early stages of the integration of a double-spanning plant viral movement protein to gain insights into how viral membrane proteins are transferred from the hydrophilic interior of the translocon into the hydrophobic environment of the bilayer, where the transmembrane (TM) segments of the viral proteins can diffuse freely. Photocrosslinking experiments reveal that this integration involves the sequential passage of the TM segments past Sec61alpha and translocating chain-associating membrane protein (TRAM). Each TM segment is first adjacent to Sec61alpha and subsequently is adjacent to TRAM. TRAM crosslinking extends for a long period during nascent chain biogenesis. In addition, the replacement of the first viral TM segment with a non-viral TM sequence still yields nascent chain photo-adducts with TRAM. TRAM therefore appears to be involved in viral membrane protein integration, and nascent chain recognition by TRAM does not appear to rely solely on the TM domains.     

WRF-TMH: predicting transmembrane helix by fusing composition index and physicochemical properties of amino acids

Membrane protein is the prime constituent of a cell, which performs a role of mediator between intra and extracellular processes. The prediction of transmembrane (TM) helix and its topology provides essential information regarding the function and structure of membrane proteins. However, prediction of TM helix and its topology is a challenging issue in bioinformatics and computational biology due to experimental complexities and lack of its established structures. Therefore, the location and orientation of TM helix segments are predicted from topogenic sequences. In this regard, we propose WRF-TMH model for effectively predicting TM helix segments. In this model, information is extracted from membrane protein sequences using compositional index and physicochemical properties. The redundant and irrelevant features are eliminated through singular value decomposition. The selected features provided by these feature extraction strategies are then fused to develop a hybrid model. Weighted random forest is adopted as a classification approach. We have used two benchmark datasets including low and high-resolution datasets. tenfold cross validation is employed to assess the performance of WRF-TMH model at different levels including per protein, per segment, and per residue. The success rates of WRF-TMH model are quite promising and are the best reported so far on the same datasets. It is observed that WRF-TMH model might play a substantial role, and will provide essential information for further structural and functional studies on membrane proteins. The accompanied web predictor is accessible at http://111.68.99.218/WRF-TMH/.     

A combination of compositional index and genetic algorithm for predicting transmembrane helical segments

Transmembrane helix (TMH) topology prediction is becoming a focal problem in bioinformatics because the structure of TM proteins is difficult to determine using experimental methods. Therefore, methods that can computationally predict the topology of helical membrane proteins are highly desirable. In this paper we introduce TMHindex, a method for detecting TMH segments using only the amino acid sequence information. Each amino acid in a protein sequence is represented by a Compositional Index, which is deduced from a combination of the difference in amino acid occurrences in TMH and non-TMH segments in training protein sequences and the amino acid composition information. Furthermore, a genetic algorithm was employed to find the optimal threshold value for the separation of TMH segments from non-TMH segments. The method successfully predicted 376 out of the 378 TMH segments in a dataset consisting of 70 test protein sequences. The sensitivity and specificity for classifying each amino acid in every protein sequence in the dataset was 0.901 and 0.865, respectively. To assess the generality of TMHindex, we also tested the approach on another standard 73-protein 3D helix dataset. TMHindex correctly predicted 91.8% of proteins based on TM segments. The level of the accuracy achieved using TMHindex in comparison to other recent approaches for predicting the topology of TM proteins is a strong argument in favor of our proposed method. Availability: The datasets, software together with supplementary materials are available at: http://faculty.uaeu.ac.ae/nzaki/TMHindex.htm.     

Membrane integration of the second transmembrane segment of band 3 requires a closely apposed preceding signal-anchor sequence

We have investigated the topogenic rules of multispanning membrane proteins using erythrocyte band 3. Here, the fine structural requirements for the correct disposition of its second transmembrane segment (TM2) were assessed. We made fusion proteins where TM1 and the loop sequence preceding TM2 were changed and fused to prolactin. They were expressed in a cell-free system supplemented with rough microsomal membrane, and their topologies on the membrane were assessed by protease sensitivity and N-glycosylation. TM1 was demonstrated to be a signal-anchor sequence that mediates translocation of the downstream portion, and thus TM2 should be responsible to halt the translocation to acquire TM topology. When the loop between TM1 and TM2 was elongated, however, TM2 was readily translocated through the membrane and not integrated. For the membrane integration of TM2, TM2 must be in close proximity to TM1. The TM1 can be replaced with another signal-anchor sequence with a long hydrophobic segment but not with a signal sequence with shorter hydrophobic stretch. The length of the hydrophobic segment affected final topology of TM2. We concluded that the two TM segments work synergistically within the translocon to acquire the correct topology and that the length of the preceding signal sequence is critical for stable transmembrane assembly of TM2. We propose that direct interaction among the TM segments is one of the critical factors for the transmembrane topogenesis of multispanning membrane proteins.     

kPROT: a knowledge-based scale for the propensity of residue orientation in transmembrane segments. Application to membrane protein structure prediction

Modeling of integral membrane proteins and the prediction of their functional sites requires the identification of transmembrane (TM) segments and the determination of their angular orientations. Hydrophobicity scales predict accurately the location of TM helices, but are less accurate in computing angular disposition. Estimating lipid-exposure propensities of the residues from statistics of solved membrane protein structures has the disadvantage of relying on relatively few proteins. As an alternative, we propose here a scale of knowledge-based Propensities for Residue Orientation in Transmembrane segments (kPROT), derived from the analysis of more than 5000 non-redundant protein sequences. We assume that residues that tend to be exposed to the membrane are more frequent in TM segments of single-span proteins, while residues that prefer to be buried in the transmembrane bundle interior are present mainly in multi-span TMs. The kPROT value for each residue is thus defined as the logarithm of the ratio of its proportions in single and multiple TM spans. The scale is refined further by defining it for three discrete sections of the TM segment; namely, extracellular, central, and intracellular. The capacity of the kPROT scale to predict angular helical orientation was compared to that of alternative methods in a benchmark test, using a diversity of multi-span alpha-helical transmembrane proteins with a solved 3D structure. kPROT yielded an average angular error of 41 degrees, significantly lower than that of alternative scales (62 degrees -68 degrees ). The new scale thus provides a useful general tool for modeling and prediction of functional residues in membrane proteins. A WWW server (http://bioinfo.weizmann.ac.il/kPROT) is available for automatic helix orientation prediction with kPROT.     

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.     

Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them.     

Transmembrane domain analysis of polycystin-1, the product of the polycystic kidney disease-1 (PKD1) gene: evidence for 11 membrane-spanning domains

Polycystin-1, the protein product of the polycystic kidney disease-1 (PKD1) gene, was originally predicted to be an integral membrane glycoprotein with 11 transmembrane (TM) domains (TM 1-11). Subsequent comparative sequence analyses led to a revision of the original model, which retained the overall topology and 11 TM segments (TM I-XI) but dropped 3 of the original domains and introduced 3 new TM domains. The membrane-spanning potential and the orientation of each of the proposed TM domains following the extracellular REJ domain (TM I-XI and TM 11) have now been tested. Using a series of N-terminal polycystin TM-glycosylation reporter gene fusions expressed in vivo, we assayed N-linked glycosylation of the C-terminal glycosylation reporter as an indicator of TM domain presence and orientation. This approach has clearly demonstrated that 7 of the 12 TM domains tested function as membrane-spanning domains. In vitro analysis of the topogenic potential of the five remaining TM domains revealed that four of these also function as membrane-spanning domains, thus supporting an 11 TM structure for polycystin-1 comprised of TM domains I-XI. In addition, these studies suggest that the membrane insertion of TM domains I-IX occurs in a cotranslational and sequential manner, while multiple topogenic determinants appear to be required for the integration of the C-terminal-most TM segments of polycystin-1.     

Membrane insertion and topology of the translocating chain-associating membrane protein (TRAM)

The translocating chain-associating membrane protein (TRAM) is a glycoprotein involved in the translocation of secreted proteins into the endoplasmic reticulum (ER) lumen and in the insertion of integral membrane proteins into the lipid bilayer. As a major step toward elucidating the structure of the functional ER translocation/insertion machinery, we have characterized the membrane integration mechanism and the transmembrane topology of TRAM using two approaches: photocross-linking and truncated C-terminal reporter tag fusions. Our data indicate that TRAM is recognized by the signal recognition particle and translocon components, and suggest a membrane topology with eight transmembrane segments, including several poorly hydrophobic segments. Furthermore, we studied the membrane insertion capacity of these poorly hydrophobic segments into the ER membrane by themselves. Finally, we confirmed the main features of the proposed membrane topology in mammalian cells expressing full-length TRAM.     

Membrane orientation of carboxyl-terminal half P-glycoprotein: topogenesis of transmembrane segments.

Multiple topological orientations of the carboxyl-terminal half of P-glycoprotein have been observed. One orientation is consistent with the hydropathy-predicted model and contains six transmembrane (TM)-spanning regions. In another orientation, the cytoplasmic-predicted loop between TM8 and TM9 is extracellular and glycosylated. In support of this "alternative" topology, TM8 was previously established to function as a signal-anchor sequence to insert with its amino-terminal end in the cytoplasm and the carboxyl-terminal end in the extracytoplasmic space. However, it is unclear how downstream TM segments fold in the membrane when TM8 functions as a signal-anchor sequence. Here, we created several chimeric Pgp molecules to examine the membrane insertion of TM segments 9 and 10 using a cell-free system. We found that TM9 functions as a stop-transfer sequence when following the signal-anchor sequence, TM8. However, the stop-transfer activity of TM9 depends on the presence of TM10. In the absence of TM10, TM9 partially translocated across the membrane into the endoplasmic reticulum lumen. In contrast, TM9 efficiently stopped the translocation event of the nascent chain in the presence of TM10. Our results suggest that the membrane insertion of TM8 and TM9 establishes the extracellular loop between TM8 and TM9. Formation of this loop apparently involves the interactions between Pgp TM segments, which facilitate proper folding of the Pgp carboxyl-terminal half.     

Dissection of De Novo Membrane Insertion Activities of Internal Transmembrane Segments of ATP-Binding-Cassette Transporters: Toward Understanding Topological Rules for Membrane Assembly of Polytopic Membrane Proteins

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an N_in-C_out rather than the predicted N_out-C_in orientation, and TM3 is in cytoplasm rather than the predicted N_in-C_out orientation in the membrane. It is possible that TM4 has a strong activity to initiate the N_in-C_out membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo N_in-C_out membrane insertion activity whereas TM4 initiates the N_in-C_out membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo N_in-C_out membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates N_in-C_out membrane insertion whereas TM4 stops the insertion event; and 2) “leaving one TM segment out of the membrane” may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the N_in-C_out membrane insertion activities of the TM segments.     

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.     

Positively charged residues are important determinants of membrane protein topology

Membrane proteins are found in a variety of conformations, with each protein spanning the membrane a set number of times and adopting a particular orientation. Positively charged residues, often located near the boundaries of transmembrane segments, appear to be involved in specifying the topology of membrane proteins.     

Differential stability of biogenesis intermediates reveals a common pathway for aquaporin-1 topological maturation

Topological studies of multi-spanning membrane proteins commonly use sequentially truncated proteins fused to a C-terminal translocation reporter to deduce transmembrane (TM) segment orientation and key biogenesis events. Because these truncated proteins represent an incomplete stage of synthesis, they transiently populate intermediate folding states that may or may not reflect topology of the mature protein. For example, in Xenopus oocytes, the aquaporin-1 (AQP1) water channel is cotranslationally directed into a four membrane-spanning intermediate, which matures into the six membrane-spanning topology at a late stage of synthesis (Skach, W. R., Shi, L. B., Calayag, M. C., Frigeri, A., Lingappa, V. R., and Verkman, A. S. (1994) J. Cell Biol. 125, 803-815 and Lu, Y., Turnbull, I. R., Bragin, A., Carveth, K., Verkman, A. S., and Skach, W. R. (2000) Mol. Biol. Cell 11, 2973-2985). The hallmark of this process is that TM3 initially acquires an Nexo/Ccyto (Type I) topology and must rotate 180 degrees to acquire its mature orientation. In contrast, recent studies in HEK-293 cells have suggested that TM3 acquires its mature topology cotranslationally without the need for reorientation (Dohke, Y., and Turner, R. J. (2002) J. Biol. Chem. 277, 15215-15219). Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.     

Topogenic properties of transmembrane segments of Arabidopsis thaliana NHX1 reveal a common topology model of the Na+/H+ exchanger family

The membrane topology of the Arabidopsis thaliana Na(+)/H(+) exchanger isoform 1 (AtNHX1) was investigated by examining the topogenic function of transmembrane (TM) segments using a cell-free system. Even though the signal peptide found in the human Na(+)/H(+) exchanger (NHE) family is missing, the N-terminal hydrophobic segment was efficiently inserted into the membrane and had an N-terminus lumen topology depending on the next TM segment. The two N-terminal TM segments had the same topology as those of TM2 and TM3 of human NHE1. In contrast, TM2 and TM3 of human NHE1 did not acquire the correct topology when the signal peptide (denoted as TM1) was deleted. Furthermore, there were three hydrophobic segments with the same topogenic properties as the TM9-H10-TM10 segments of human NHE1, which has one lumenal loop (H10) and two flanking TM segments (TM9 and TM10). These data indicate that the plant NHX isoforms can form the common membrane topology proposed for the human NHE family, even though it does not have a signal peptide.     

Orientation of Small Multidrug Resistance Transporter Subunits in the Membrane: Correlation with the Positive-Inside Rule

Small multidrug resistance (SMR) transport proteins provide a model for the evolution of larger two-domain transport proteins. The orientation in the membrane of 27 proteins from the SMR family was determined using the reporter fusion technique. Nine members were encoded monocistronically (singles) and shown to insert in both orientations (dual topology). Eighteen members were encoded in pairs on the chromosome and shown to insert in fixed orientations; the two proteins in each pair invariably had opposite orientations in the membrane. Interaction between the two proteins in pairs was demonstrated by copurification. The orientation in the membrane of either protein in the pair was affected only marginally by the presence of the other protein.For the proteins in pairs, the orientation in the membrane correlated well with the distribution of positively charges residues (R + K) over the cytoplasmic and extracellular loops (positive-inside rule). In contrast, dual-topology insertion of the singles was predicted less well by the positive-inside rule. Three singles were predicted to insert in a single orientation with the N-terminus and the C-terminus at the extracellular side of the membrane. Analysis of charge distributions suggests the requirement of a threshold number of charges in the cytoplasmic loops for the positive-inside rule to be of predictive value. It is concluded that a combined analysis of gene organization on the chromosome and phylogeny is sufficient to distinguish between fixed or dual topology of SMR members and, probably, similar types of membrane proteins. The positive-inside rule can be used to predict the orientation of members in pairs, but is not suitable as a sole predictor of dual topology.     

Converting a marginally hydrophobic soluble protein into a membrane protein

δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.