Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context

Hedin, Linnea E; Öjemalm, Karin; Bernsel, Andreas; Hennerdal, Aron; Illergård, Kristoffer; Enquist, Karl; Kauko, Anni; Cristóbal, Susana; Heijne, Gunnar von; Lerch-Bader, Mirjam; Nilsson, IngMarie; Elofsson, Arne

doi:10.1016/j.jmb.2009.11.036

Cited by 88 publications

(134 citation statements)

References 24 publications

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“…Translation products were analyzed by SDS-PAGE. Gels were visualized on a Fuji FLA-3000 PhosphoImager using the Image Reader 8.1j software and quantified using ImageGauge V 3.45 and the Qtiplot 0.9.3-rc2 softwares (35). The degree of membrane integration of each H segment was calculated as an apparent equilibrium constant between the membrane-integrated and nonintegrated forms: K app ¼ f 1 ∕f 2 , where f 1 is the fraction of singly and f 2 the fraction of doubly glycosylated Lep molecules, and the results were then converted to apparent free energies, ΔG app ¼ −RT ln K app .…”

Section: Methodsmentioning

confidence: 99%

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

Öjemalm

Higuchi

Jiang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid's contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal∕ðmol · Å 2 Þ, the solvation parameter for transfer from translocon to bilayer is 6-10 cal∕ðmol · Å 2 Þ, pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon.flexizyme | hydrophobicity | nonproteinogenic amino acid | transmembrane helix I n eukaryotic cells, membrane proteins destined for the plasma membrane and the various compartments along the endo-and exocytic pathways are synthesized by endoplasmic reticulum (ER)-bound ribosomes and cotranslationally integrated into the ER membrane in a process mediated by the Sec61 translocon complex; the homologous SecYEG translocon mediates membrane-protein integration into the inner membrane of prokaryotes (1, 2). Subsequent to membrane integration, membrane proteins fold and oligomerize in the ER and are then moved further along the secretory pathway by vesicular transport.During the membrane-integration step, hydrophobic segments in the translocating nascent polypeptide chain exit the Sec61 translocon through a lateral gate and become embedded in the surrounding lipid bilayer (3-5). Cotranslational, transloconmediated integration of transmembrane α-helices into the ER membrane sets the stage for all subsequent folding and oligomerization events and hence represents a critical step in the maturation of membrane proteins.In previous studies, we have provided quantitative data on the propensities of the 20 natural amino acids to promote the integration of transmembrane helices into the ER membrane and have shown that they depend both on hydrophobicity and on position within the helix (3, 6). Although the partitioning of transmembrane helices between the Sec61 translocon and the lipid membrane bears strong similarities to partitioning of solutes between water and lipid membranes, translocon-to-bilayer partitioning may not be equivalent to water-to-bilayer partitioning (7). Insights into the differences between the two partitioning processes might be revealed i...

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Section: Methodsmentioning

confidence: 99%

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

Öjemalm

Higuchi

Jiang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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confidence: 99%

“…Although hydrophobicity is the predominant factor (16), it is insufficient per se to determine whether or not a given polypeptide segment will be integrated into the membrane (17,18). Both residues immediately flanking a transmembrane (TM) segment (19) and those in other TM segments upstream or downstream (18,20) can also contribute to the membrane insertion propensity of the segment. Experiments have demonstrated that the polypeptide chain can interact with lipids while still in the channel (21)(22)(23), leading to the proposal that insertion is a thermodynamic partitioning between channel and membrane (23)(24)(25).…”

mentioning

confidence: 99%

Free-energy cost for translocon-assisted insertion of membrane proteins

Gumbart

Chipot

Schulten

2011

Proc. Natl. Acad. Sci. U.S.A.

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Nascent membrane proteins typically insert in a sequential fashion into the membrane via a protein-conducting channel, the Sec translocon. How this process occurs is still unclear, although a thermodynamic partitioning between the channel and the membrane environment has been proposed. Experiment-and simulationbased scales for the insertion free energy of various amino acids are, however, at variance, the former appearing to lie in a narrower range than the latter. Membrane insertion of arginine, for instance, requires 14-17 kcal∕mol according to molecular dynamics simulations, but only 2-3 kcal∕mol according to experiment. We suggest that this disagreement is resolved by assuming a two-stage insertion process wherein the first step, the insertion into the translocon, is energized by protein synthesis and, therefore, has an effectively zero free-energy cost; the second step, the insertion into the membrane, invokes the translocon as an intermediary between the fully hydrated and the fully inserted locations. Using free-energy perturbation calculations, the effective transfer free energies from the translocon to the membrane have been determined for both arginine and leucine amino acids carried by a background polyleucine helix. Indeed, the insertion penalty for arginine as well as the insertion gain for leucine from the translocon to the membrane is found to be significantly reduced compared to direct insertion from water, resulting in the same compression as observed in the experiment-based scale.membrane-protein insertion | SecY | ribosome | hydrophobicity scale N early all membrane proteins found in the inner membranes of bacterial cells and the membranes of eukaryotic cells are inserted concomitant with their synthesis by the ribosome, i.e., cotranslationally (1). Insertion into the bilayer does not happen directly, but rather occurs via a highly conserved protein-conducting channel in the membrane, the Sec translocon (2-4). At an early stage of synthesis, the ribosome docks to the channel, forming a tightly bound complex (5, 6). The polypeptide is then inserted into the translocon prior to entering the membrane, the former step requiring a driving force, such as nucleotide hydrolysis, a membrane potential gradient, or the pressure exerted by the growing nascent chain (2).In addition to aiding the insertion of membrane proteins, the translocon also allows certain nascent proteins to cross the membrane (2). Structures of the Sec translocon (7-9) reveal two apparent gates, one transverse for the passage of soluble proteins across the membrane and one lateral for the exit of membrane proteins (7,10,11). The lateral gate is formed at the interface of two halves of SecY (7, 12) (see Fig. 1B). This gate fluctuates during translocation of the nascent polypeptide (13,14), although what factors govern its opening and closing are unclear (8,9,15).Given the two pathways presented by the translocon, there must exist a way to discriminate between proteins destined for the lipid membrane environment and proteins destined ...

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“…1a). It has been shown previously that, in some cases, a neighboring TM helix can promote membrane insertion of a marginally hydrophobic TM region [16][17][18][19] and that there is a correlation between the polarity of a TM helix and its interaction area with the rest of the protein. 20 Therefore, we investigated the insertion of the full α-helical hairpin region (residues 35-81) in this in vitro translation system.…”

Section: Insertion Of the Viroporin 2b Hairpin Region Into Biologicalmentioning

confidence: 99%

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

Bañó‐Polo

Martínez-Gil

Wallner

et al. 2013

Journal of Molecular Biology

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α-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM-TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures.

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Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context

Cited by 88 publications

References 24 publications

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

Free-energy cost for translocon-assisted insertion of membrane proteins

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

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