Positive-inside Rule Research Articles

Spf1 and Ste24: quality controllers of transmembrane protein topology in the eukaryotic cell.

DNA replication, transcription, and translation in eukaryotic cells occur with decreasing but still high fidelity. In contrast, for the estimated 33% of the human proteome that is inserted as transmembrane (TM) proteins, insertion with a non-functional inverted topology is frequent. Correct topology is essential for function and trafficking to appropriate cellular compartments and is controlled principally by responses to charged residues within 15 residues of the inserted TM domain (TMD); the flank with the higher positive charge remains in the cytosol (inside), following the positive inside rule (PIR). Yeast (Saccharomyces cerevisiae) mutants that increase insertion contrary to the PIR were selected. Mutants with strong phenotypes were found only in SPF1 and STE24 (human cell orthologs are ATP13A1 and ZMPSte24) with, at the time, no known relevant functions. Spf1/Atp13A1 is now known to dislocate to the cytosol TM proteins inserted contrary to the PIR, allowing energy-conserving reinsertion. We hypothesize that Spf1 and Ste24 both recognize the short, positively charged ER luminal peptides of TM proteins inserted contrary to the PIR, accepting these peptides into their large membrane-spanning, water-filled cavities through interaction with their many interior surface negative charges. While entry was demonstrated for Spf1, no published evidence directly demonstrates substrate entry to the Ste24 cavity, internal access to its zinc metalloprotease (ZMP) site, or active withdrawal of fragments, which may be essential for function. Spf1 and Ste24 comprise a PIR quality control system that is conserved in all eukaryotes and presumably evolved in prokaryotic progenitors as they gained differentiated membrane functions. About 75% of the PIR is imposed by this quality control system, which joins the UPR, ERAD, and autophagy (ER-phagy) in coordinated, overlapping quality control of ER protein function.

Analysis of Protein-Cardiolipin Interactions in the E. coli Inner Membrane

Integral membrane proteins are localised and functionally regulated by lipids present in the surrounding bilayer. Whilst bacteria such as E. coli have a relatively simple membrane when compared to eukaryotic cells, there is ample evidence that multiple proteins bind, and have their function regulated by, specific lipid interactions. There are a number of ways of identifying protein-lipid interactions in membrane proteins, including through molecular simulation. Here, we highlight the capabilities of this technique by running lipid-binding simulations of 42 different E. coli inner membrane proteins. Our data reveals a strong cross-membrane asymmetry in the nature of protein-lipid interactions, with a marked increase of anionic lipid binding to the inner leaflet regions of membrane proteins. This appears to be driven by an increase in basic residues, suggesting a structural basis for the positive inside rule. We have identified ca. 700 independent cardiolipin binding sites from our data, allowing us dissect the molecular basis of a prototypical cardiolipin headgroup binding site. Applying free energy calculations quantifies the relative contributions of each residue type to cardiolipin binding. These findings demonstrate the potential for large-scale simulations to reveal pertinent underlying biological information applying to a number of systems, and paves the way for future analyses of more complex protein-lipid interactions.

Lipid-Assisted Membrane Protein Folding and Topogenesis.

Due to the heterogenous lipid environment in which integral membrane proteins are embedded, they should follow a set of assembly rules, which govern transmembrane protein folding and topogenesis accordingly to a given lipid profile. Recombinant strains of bacteria have been engineered to have different membrane phospholipid compositions by molecular genetic manipulation of endogenous and foreign genes encoding lipid biosynthetic enzymes. Such strains provide a means to investigate the in vivo role of lipids in many different aspects of membrane function, folding and biogenesis. In vitro and in vivo studies established a function of lipids as molecular chaperones and topological determinants specifically assisting folding and topogenesis of membrane proteins. These results led to the extension of the Positive Inside Rule to Charge Balance Rule, which incorporates a role for lipid-protein interactions in determining membrane protein topological organization at the time of initial membrane insertion and dynamically after initial assembly. Membrane protein topogenesis appears to be a thermodynamically driven process in which lipid-protein interactions affect the potency of charged amino acid residues as topological signals. Dual topology for a membrane protein can be established during initial assembly where folding intermediates in multiple topological conformations are in rapid equilibrium (thus separated by a low activation energy), which is determined by the lipid environment. Post-assembly changes in lipid composition or post-translational modifications can trigger a reorganization of protein topology by inducing destabilization and refolding of a membrane protein. The lipid-dependent dynamic nature of membrane protein organization provides a novel means of regulating protein function.

Membrane Protein Integration and Topogenesis at the ER.

Most membrane proteins are composed of hydrophobic α-helical transmembrane segments and are integrated into the lipid bilayer of the endoplasmic reticulum by the highly conserved Sec61 translocon. With respect to the integration mechanism, three types of transmembrane segments can be distinguished-the signal, the stop-transfer sequence, and the re-integration sequence-which in linear succession can account for all kinds of membrane protein topologies. The transmembrane orientation of the initial signal and to a weaker extent also of downstream transmembrane segments is affected by charged flanking residues according to the so-called positive-inside rule. The main driving force for transmembrane integration is hydrophobicity. Systematic analysis suggested thermodynamic equilibration of each peptide segment in the translocon with the membrane as the underlying mechanism. However, there is evidence that integration is not entirely sequence-autonomous, but depends also on the sequence context, from very closely spaced transmembrane segments to the folding state and properties of neighboring sequences. Topogenesis is even influenced by accessory proteins that appear to act as intramembrane chaperones.

Charged residues next to transmembrane regions revisited: \u201cPositive-inside rule\u201d is complemented by the \u201cnegative inside depletion/outside enrichment rule\u201d

BackgroundTransmembrane helices (TMHs) frequently occur amongst protein architectures as means for proteins to attach to or embed into biological membranes. Physical constraints such as the membrane’s hydrophobicity and electrostatic potential apply uniform requirements to TMHs and their flanking regions; consequently, they are mirrored in their sequence patterns (in addition to TMHs being a span of generally hydrophobic residues) on top of variations enforced by the specific protein’s biological functions.ResultsWith statistics derived from a large body of protein sequences, we demonstrate that, in addition to the positive charge preference at the cytoplasmic inside (positive-inside rule), negatively charged residues preferentially occur or are even enriched at the non-cytoplasmic flank or, at least, they are suppressed at the cytoplasmic flank (negative-not-inside/negative-outside (NNI/NO) rule). As negative residues are generally rare within or near TMHs, the statistical significance is sensitive with regard to details of TMH alignment and residue frequency normalisation and also to dataset size; therefore, this trend was obscured in previous work. We observe variations amongst taxa as well as for organelles along the secretory pathway. The effect is most pronounced for TMHs from single-pass transmembrane (bitopic) proteins compared to those with multiple TMHs (polytopic proteins) and especially for the class of simple TMHs that evolved for the sole role as membrane anchors.ConclusionsThe charged-residue flank bias is only one of the TMH sequence features with a role in the anchorage mechanisms, others apparently being the leucine intra-helix propensity skew towards the cytoplasmic side, tryptophan flanking as well as the cysteine and tyrosine inside preference. These observations will stimulate new prediction methods for TMHs and protein topology from a sequence as well as new engineering designs for artificial membrane proteins.

Effects of mixed proximal and distal topogenic signals on the topological sensitivity of a membrane protein to the lipid environment

The final topology of membrane proteins is thought to be dictated primarily by the encoding sequence. However, according to the Charge Balance Rule the topogenic signals within nascent membrane proteins are interpreted in agreement with the Positive Inside Rule as influenced by the protein phospholipid environment. The role of long-range protein-lipid interactions in establishing a final uniform or dual topology is unknown. In order to address this role, we determined the positional dependence of the potency of charged residues as topological signals within Escherichia coli sucrose permease (CscB) in cells in which the zwitterionic phospholipid phosphatidylethanolamine (PE), acting as topological determinant, was either eliminated or tightly titrated. Although the position of a single or paired oppositely charged amino acid residues within an extramembrane domain (EMD), either proximal, central or distal to a transmembrane domain (TMD) end, does not appear to be important, the oppositely charged residues exert their topogenic effects separately only in the absence of PE. Thus, the Charge Balance Rule can be executed in a retrograde manner from any cytoplasmic EMD or any residue within an EMD most likely outside of the translocon. Moreover, CscB is inserted into the membrane in two opposite orientations at different ratios with the native orientation proportional to the mol % of PE. The results demonstrate how the cooperative contribution of lipid-protein interactions affects the potency of charged residues as topological signals, providing a molecular mechanism for the realization of single, equal or different amounts of oppositely oriented protein within the same membrane.

Statistical Models Robustly Predict Membrane Protein Expression in E. Coli

Membrane protein production is difficult; their biogenesis does not stop with translation but also requires translocation and integration into a lipid bilayer. These additional steps hamper their heterologous expression which significantly impedes biophysical and structural studies. Detailed and anecdotal evidence in the literature suggests that a variety nucleotide and amino-acid sequence level determinants may potentially support or hinder their biogenesis, e.g. mRNA pausing elements, codon adaptation, transmembrane segment hydrophobicity, “positive inside rule.” In previous work, we demonstrated that a linear, preference-ranking Support Vector Machine can capture heterologous membrane protein expression in E. coli. Here we present work showing statistical models with greatly improved performance across small- and large-scale laboratory experiments (e.g. expression tests that routinely precede structural studies) published in the literature. We also present progress towards a Bayesian model that can be refined as new expression data becomes available on-line and that can be extended across expression systems (e.g. yeast, insect cells), plasmids, and conditions. Furthermore, parameter values from this model will facilitate reliably characterizing the sequence features underpinning membrane protein expression suggesting intriguing areas for further biophysical and computational experiments.

Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology

The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTβL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTβL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is -6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (topgraph.weizmann.ac.il).

Making dimers of oligomeric membrane proteins using copper-free click chemistry

Here we describe the development of a protocol to make small oligomers, dimers and trimers, from highly oligomeric membrane proteins. The proteins that we used are the light harvesting 2 proteins and core complexes from photosynthetic bacteria, which contain respectively 16 and 56 individual polypeptides. Creating specific dimers between such multimeric protein poses several problems. We propose a protocol based on asymmetric lysine localization, thanks to the positive inside rule, and copper-free click chemistry. With this method we are able to produce specific dimeric complexes in detergent solution of possible biological relevance.

Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

A Machine Learning Approach to Heterologous Membrane Protein Overexpression

Membrane protein production is difficult; their biogenesis does not stop with translation but also requires translocation and integration into a lipid bilayer. These additional steps hamper their heterologous expression which significantly impedes biophysical and structural studies. Detailed and anecdotal evidence in literature suggests that a variety nucleotide and amino-acid sequence level determinants may potentially support or hinder their biogenesis, e.g. mRNA pausing elements, codon adaptation, transmembrane segment hydrophobicity, “positive inside rule.” By training a preference-ranking Support Vector Machine, we have developed a statistical model that predicts a relative likelihood of a membrane protein's successful expression using quantitative experimental data of overexpression. This model is rigorously validated against expression outcomes from small-scale laboratory experiments (e.g. expression tests that routinely precede structural studies) published in the literature as well as large-scale expression trials from a Protein Structure Initiative consortium facility. We show remarkable agreement between the predicted and experimental expression outcomes and propose our model, trained and cross-validated on the entire corpus of data, as a tool for the membrane protein biophysics community to streamline the process of overexpressing a target for study. Given the framework of our model, it can be trivially re-trained as additional experimental outcomes are gathered from past work or created from experiments. Furthermore, the relative weights gathered from parameters of the statistical model may help further characterize translocation mechanisms and suggest intriguing areas for further biophysical and computational experiments.

Membrane Protein Misfolding Enforces the Positive-Inside Rule

Inner membrane proteins have long been known to follow the “positive-inside rule”, where cytoplasmic loops tend to have a greater number of cationic residues than periplasmic or extracellular loops. This effect can be seen dramatically in dual-topology proteins, such as the small multidrug transporter EmrE, where relatively balanced charge between the protein faces leads to mixed insertion in the membrane; some molecules are inserted with a cytoplasmic N-terminus, and some with a periplasmic. Addition or removal of positive charge can bias the orientation of the inserted protein. Surprisingly, this is true even if the mutation does not occur until after the synthesis of several transmembrane helices. Here, we examine how positive charges bring about orientation bias. We use a GFP-based assay to examine both the orientation of the protein as well as rates of misinsertion and degradation. We find a significant pool of degraded or misfolded protein to be present even with wild-type EmrE. In addition to the redistribution of well-folded protein, the amount, type, and orientation of degradation products changes dramatically upon the introduction of mutations that bias EmrE orientation. While early mutations, especially those surrounding the first transmembrane helix, appear capable of biasing the initial orientation, the effects of later mutations can be explained by altering the rate of misfolding events in accordance with the positive-inside rule. Positive charges thus appear to act as folding signals, enhancing proper folding and insertion when positioned in agreement with the positive-inside rule, and enhancing misfolding when placed in violation.

Electrostatics inside the SecY Translocon

SecY constitutes the core of the highly conserved SecYEG translocon complex that governs the translocation or integration of proteins across or into cytoplasmic membranes. Besides its role of guiding nascent membrane protein chains into the lipid bilayer, it also plays an important role in determining transmembrane topology via the so-called positive-inside rule. Site-directed mutations of translocons are known to modify the topology of nascent α-helical segments [1]. We performed molecular dynamics simulations of SecYE from Pyrococcus furiosus [2] embedded in a POPC bilayer. In this structure, SecY is in a partially open ‘primed’ state. It conveniently remained open over the course of a 450 ns simulation due to the presence of lipids wedged between helices TM2b and TM7 (the lateral gate), thus allowing close examination of the electrostatics within the translocon. Within the channel, water dipoles exhibit a preferred orientation, which affects the electrostatic (ES) potential. Time-averaged ES maps reveal a largely positive potential relative to the bulk aqueous phase within the channel except for a strong negative peak at the N-terminus of TM2b. This has implications for the entry of signal sequences into the translocon channel.Research supported by NIH grants RO1 GM74637 and PO1 GM86685.

The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix

The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.

Protein Gymnastics in Thelipid Bilayer: Lipides as Determinants of Protein Structure

The orientation of transmembrane domains of polytopic membrane proteins with respect to the plane of the lipid bilayer is determined by a complex interplay between topogenic signals residing within the protein sequence, interaction of the protein with the translocon membrane insertion machinery, short-range and long-range interactions within the protein and the final environment of the protein largely determined by membrane lipid composition. Systematic alteration of lipid composition at steady state or dynamically after membrane protein assembly uncovered a role for lipid-protein interactions in determining initial membrane protein topogenesis as well as in dynamic topological re-organization after initial protein folding. Alteration of the charges within protein extramembrane domains coupled with changes in lipid composition demonstrated a synergistic and reciprocal relationship between protein and lipid charges in determining orientation of transmembrane domains. These results led to the Charge Balance Hypothesis, which posits that protein topogenic signals are decoded in accordance with positive-inside rule initially by the translocon but finally interpreted by electrostatic interactions between protein extramembrane domains and the membrane surface charge as determined by the collective membrane lipid head group composition. The steady state and dynamic nature of membrane protein organization observed in whole cells, as a function of the Charge Balance Hypothesis, has been faithfully reproduced with membrane proteins reconstituted in proteoliposomes thus establishing that such initial and dynamic protein organization is dependent solely on direct lipid-protein interactions independent of other cellular factors. Therefore, membrane protein topological organization can be viewed as highly dynamic rather than stable and static. The dynamic view of protein topological organization as influenced by the membrane lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded membrane proteins. Supported in part by NIH grant R37-GM20478.

Positive Charge Loading at Protein Termini Is Due to Membrane Protein Topology, Not a Translational Ramp

In the great majority of genomes, the use of positive charge increases, on average, approaching protein N-termini. Such charged residues slow ribosomes by interacting with the negatively charged exit tunnel. This has been proposed to be selectively advantageous as it provides an elongation speed ramp at translational starts. Positive charges, however, are known to orientate proteins in membranes by the positive-inside rule whereby excess charge lies on the cytoplasmic side of the membrane. Which of these two models better explains the N-terminal loading of positively charged amino acids? We find strong evidence that the tendency for average positive charge use to increase at termini is exclusively due to membrane protein topology: 1) increasing N-terminal positive charge is not found in cytosolic proteins, but in transmembrane ones with cytosolic N-termini, with signal sequences contributing additional charge; 2) positive charge density at N-termini corresponds to the length of cytoplasmically exposed transmembrane tails, its usage increasing just up until the membrane; 3) membrane-related patterns are repeated at C-termini, where no ramp is expected; and 4) N-terminal positive charge patterns are no different from those seen internally in proteins in membrane-associated domains. The overall apparent increase in positive charge across all N-termini results from membrane proteins using positive charge adjacent to the cytosolic leaflet, combined with a skewed distribution of where N-termini cross the plasma membrane; 5) while Escherichia coli was predicted to have a 5' ribosomal occupancy ramp of at least 31 codons, in contrast to what is seen in yeast, we find in ribosomal footprinting data no evidence for such a ramp. In sum, we find no need to invoke a translational ramp to explain the rising positive charge densities at N-termini. The membrane orientation model makes a full account of the trend.

Insertion Mechanism in E. Coli of Single-Span Membrane Proteins with Far-Downstream Transmembrane Segments

Single-span membrane proteins (SSMPs) are the most abundant membrane proteins in virtually all organisms. SSMPs with N-terminus outside the cell are referred to as Type I MPs, while those with the N-terminus inside are referred to as Type II MPs. It is generally assumed that insertion occurs co-translationally via the signal recognition particle (SRP) pathway. The positive-inside rule determines TypeI/II MPs. The TM segment of Type I MPs is generally preceded by a cleavable signal sequence to facilitate the Nout-Cin topology. The TM segment of both types of proteins generally occurs early in the amino acid sequence, allowing recognition by SRP as it emerges from the ribosome. Secreted periplasmic proteins have a signal sequence, but are secreted post-translationally through the translocon by the SecA translocase.We are studying an unusual class of Type II MPs that lack identifiable signal sequences and whose TM segments can occur hundreds of residues downstream from the N-terminus. One such protein is RodZ, which is a cytoskeletal protein involved in maintaining the rod shape of E. coli. What pathway does this protein use? Several prediction programs and our experimental studies of RodZ with deleted TM segment (RodZ-ΔTM) confirm the absence of a native targeting signal. Proteinase K treatment of spheroplasts reveal that RodZ has Nin-Cout (Type II) topology, consistent with previous studies. Classic N-terminal cleavable signal sequences, such as DsbA or MalE, do not affect the topology of RodZ. We have determined that RodZ insertion is strongly SecA dependent, because RodZ is not incorporated into the membrane under SecA-depletion conditions or in the presence of sodium azide, which is known to inhibit SecA. This result is important, because it is now possible to study the energetics of TM helix insertion by SecA.

Role of Individual Positive Charges in the Membrane Orientation and Activity of Transporters of the Small Multidrug Resistance Family

The effect of individual positively charged residues on the orientation in the membrane was analyzed in three dual-topology transporters of the small multidrug resistance (SMR) family: AAVE4701aave of Acidovorax avenae, EMREecol of Escherichia coli, and RRUA0272rrub of Rhodospirillum rubrum. It is shown that (i) individual positive charges have different impacts on the orientation, (ii) positive charges that are conserved in the three different proteins do not have the same impact on the orientation, (iii) positive charges in odd- and even-numbered loops have different impacts, (iv) for some, but not all, the impact depends on the presence of other positive charges, and (v) proteins from which all positive charges are removed in some cases are dual-topology proteins and in other cases have a single orientation. A small number of positive charges placed in the loops of the latter proteins results in the violation of the so-called positive-inside rule that has been reported previously [Kolbusz, M. A., et al. (2010) J. Mol. Biol. 402, 127-138]. We conclude that each positive charge shifts the distribution between the two orientations toward the state that has the positive charge in the cytoplasm but that intrinsic factors other than positive charges determine the orientation as well. The ability of the mutants of AAVE4701aave and EMREecol to confer resistance against ethidium bromide revealed an essential role in catalysis for a conserved pair of positive charges in the second loop. No significant relation between activity and the relative orientation of the monomeric subunits in the dimer could be demonstrated.

Orientation of Internal Signal-Anchor Sequences at the Sec61 Translocon

Translocation and insertion of secretory and membrane proteins at the endoplasmic reticulum are mediated by the Sec61 translocon. Evidence from in vivo as well as in vitro experiments indicates that N-terminal signal-anchor sequences initially insert N-first before they invert their orientation to translocate the C-terminus. Inversion is driven by flanking charges according to the positive-inside rule and inhibited by increased signal hydrophobicity. Here, we show that upon extending the N-terminal hydrophilic domain preceding the signal core to more than ~20 residues, the insertion behavior changes. Apparent signal inversion and the effect of hydrophobicity are largely lost, suggesting that N-first insertion is limited to N-terminal signal anchors. Extended N-domains sterically hinder N-translocation in a length-dependent manner also for reverse signal anchors with inverted flanking charges. The results indicate a mechanistic difference in the insertion process of N-terminal and internal signal sequences.

Membrane composition influences the topology bias of bacterial integral membrane proteins

Small multidrug resistance (SMR) protein family members confer bacterial resistance to toxic antiseptics and are believed to function as dual topology oligomers. If dual topology is essential for SMR activity, then the topology bias should change as bacterial membrane lipid compositions alter to maintain a “neutral” topology bias. To test this hypothesis, a bioinformatic analysis of bacterial SMR protein sequences was performed to determine a membrane protein topology based on charged amino acid residues within loops, and termini regions according to the positive inside rule. Three bacterial lipid membrane parameters were examined, providing the proportion of polar lipid head group charges at the membrane surface (PLH), the relative hydrophobic fatty acid length (FAL), and the proportion of fatty acid unsaturation (FAU). Our analysis indicates that individual SMR pairs, and to a lesser extent SMR singleton topology biases, are significantly correlated to increasing PLH, FAL and FAU differences validating the hypothesis. Correlations between the topology biases of SMR proteins identified in Gram+ compared to Gram− species and each lipid parameter demonstrated a linear inverse relationship.