Positively and negatively charged residues have different effects on the position in the membrane of a model transmembrane helix

Monné, Magnus; Nilsson, IngMarie; Johansson, Mikael P.; Elmhed, N.; Heijne, Gunnar von

doi:10.1006/jmbi.1998.2218

Cited by 101 publications

(115 citation statements)

References 20 publications

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“…Using in vitro translation of model proteins in the presence of RMs, we show that a run of 3-4 Ala residues placed centrally in the poly-Leu stretch efficiently induces helical hairpin formation, thus suggesting that Ala, at least in this context, has a slight preference for the lipid-water interface region over the central acyl chain region. As additional support for this contention, we show that a transmembrane segment composed of 13 Leu followed by 10 Ala and one Val is partly cleaved by signal peptidase near its C terminus, that cleavage is prevented by the addition of a Leu residue to the C-terminal end of the Ala-stretch, and that a part of the Ala-stretch appears to extend well into the lipid-water interface region as determined by the glycosylation mapping technique (16). These results agree surprisingly well with recent biophysical studies on simple peptide-lipid systems (17), suggesting that the nascent polypeptide chain can interact with membrane lipids at a very early stage of the translocon-mediated insertion of membrane proteins into the ER.…”

supporting

confidence: 54%

How Hydrophobic Is Alanine?

Nilsson

Johnson

Heijne

2003

Journal of Biological Chemistry

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By a number of measures, alanine is poised at the threshold between those amino acids that promote the membrane integration of transmembrane ␣-helices and those that do not. We have measured the preference of alanine to partition into the lipid-water interface region over the central acyl chain region of the endoplasmic reticulum (ER) membrane both by its ability to promote the formation of so-called helical hairpins, i.e. a pair of transmembrane helices separated by a tight turn, and by mapping the position relative to the membrane of the lumenal end of a transmembrane ␣-helix that ends with a block of 10 alanines. Both measures show that Ala has a weak but distinct preference for the interface region, which is in agreement with recent biophysical measurements on pentaeptide partitioning in simple water-lipid or water-octanol systems (Jayasinghe, S., Hristova, K., and White, S. H. (2001) J. Mol. Biol. 312, 927-934). Considering the complexity of the translocon-mediated insertion of membrane proteins into the ER, the agreement between the biochemical and biophysical measurements is striking and suggests that proteinlipid interactions are already important during the very early steps of membrane protein assembly in the ER.Most integral membrane proteins are composed of tightly packed bundles of transmembrane ␣-helices (1). The loops connecting the helices tend to be short (2), suggesting the possibility that pairs of helices with a short connecting loop, i.e. a helical hairpin (3), may be inserted en bloc into the membrane (4 -6) rather than the helices being recognized and inserted one by one by the translocation/membrane insertion machinery (7).Using in vitro translation in the presence of rough microsomes (RMs) 1 of model membrane proteins containing engineered poly-Leu segments of various lengths, we have shown previously that the balance between forming a long, continuous transmembrane helix and a helical hairpin can be a delicate one; in constructs with 35-40-residue-long poly-Leu stretches, a single Leu 3 Pro mutation near the middle of the poly-Leu stretch is sufficient to convert a long transmembrane helix into a helical hairpin (8, 9). Other polar and charged residues also promote helical hairpin formation in this context (9 -11). As an example of a similar effect in a wild type protein, an Asn-Pro 3 Leu-Leu mutation between two tightly spaced transmembrane helices in the Saccharomyces cerevisiae Ste14p protein has been shown to convert the helical hairpin into a single, long transmembrane helix (12).In our previous studies (9, 10), Ala has not shown any tendency to promote the formation of helical hairpins. The question of whether Ala should be considered a "membrane-seeking" or a "water-seeking" residue is not an easy one, however, as Ala balances close to the hydrophobic/hydrophilic threshold in many well known hydrophobicity scales (13). Poly-Ala segments of ϳ20 residues or more can insert as transmembrane helices during membrane protein assembly into the endoplasmic reticulum (ER) membrane (14), bu...

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confidence: 54%

How Hydrophobic Is Alanine?

Nilsson

Johnson

Heijne

2003

Journal of Biological Chemistry

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“…2F, anti-Spep). Positively charged residues have been reported to be accommodated in other naturally occurring and model transmembrane helices (34,46). Interestingly, SSP association in GP-C was not completely abrogated by the F46K and F49K mutations and could be detected in overly darkened images from Fig.…”

mentioning

confidence: 87%

Bitopic Membrane Topology of the Stable Signal Peptide in the Tripartite Junín Virus GP-C Envelope Glycoprotein Complex

Agnihothram

York²,

Trahey³

et al. 2007

J Virol

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The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (h1 and h2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning h2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex.

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“…It is therefore plausible that during CFTR synthesis, the relatively polar nature of wild type TM8 favors an unstructured conformation as the nascent chain enters the translocon lumen. This would extend TM8 N-terminal residues ϳ30 Å further into the ER lumen than would a helical conformation and transiently increase accessibility to OST (55,60,61). In this model, residue Asp 924 would be expected to delay membrane integration (and possibly helix formation) by decreasing the tendency of the helix to move into the bilayer through the lateral translocon gate (23).…”

Section: Figmentioning

confidence: 99%

Cooperativity and Flexibility of Cystic Fibrosis Transmembrane Conductance Regulator Transmembrane Segments Participate in Membrane Localization of a Charged Residue

Carveth

Buck

Anthony

et al. 2002

Journal of Biological Chemistry

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Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp 924 , within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis.The topology of most eukaryotic polytopic proteins is generated in the endoplasmic reticulum (ER) 1 through the collective action of sequence determinants encoded within the nascent polypeptide. These determinants encompass hydrophobic transmembrane (TM) segments that, together with their flanking residues, interact with cytosolic and ER translocation machinery to initiate and terminate translocation and integrate the polypeptide into the lipid bilayer (reviewed in Refs. 1-3). In the simplest model, topology can be established cotranslationally by alternating topogenic determinants that function as signal (anchor) and stop transfer sequences (4 -7). As the first signal sequence emerges from the ribosome, it targets the ribosome nascent-chain complex (RNC) to the ER and gates open an aqueous channel in the membrane (the Sec61 translocon) (8). Because the ribosome exit site is directly aligned with the axial pore of the translocon, newly synthesized polypeptide is cotranslationally directed into the aqueous environment of the translocon as it emerges from the ribosome (9 -11). Subsequent synthesis of a stop transfer sequence gates the translocon closed to the ER lumen, terminates translocation, and provides the growing nascent polypeptide access to the cytosol (12, 13). Through sequential iterations of these events, signal and stop transfer sequences can alternately direct the polypeptide into the ER lumen or the cytosol and thus establish topology of transmembrane segments and lumenal and cytosolic peptide loops.Not all native polytopic proteins utilize a simple cotranslational biogenesis pathway. For ex...

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Positively and negatively charged residues have different effects on the position in the membrane of a model transmembrane helix

Cited by 101 publications

References 20 publications

How Hydrophobic Is Alanine?

How Hydrophobic Is Alanine?

Bitopic Membrane Topology of the Stable Signal Peptide in the Tripartite Junín Virus GP-C Envelope Glycoprotein Complex

Cooperativity and Flexibility of Cystic Fibrosis Transmembrane Conductance Regulator Transmembrane Segments Participate in Membrane Localization of a Charged Residue

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