What sequence features in integral membrane proteins determine which parts of the polypeptide chain will form transmembrane alpha-helices and which parts will be located outside the lipid bilayer? Previous studies on the integration of model transmembrane segments into the mammalian endoplasmic reticulum (ER) have provided a rather detailed quantitative picture of the relation between amino acid sequence and membrane-integration propensity for proteins targeted to the Sec61 translocon. We have now carried out a comparative study of the integration of N out-C in-orientated 19-residue-long polypeptide segments into the ER of the yeast Saccharomyces cerevisiae. We find that the 'threshold hydrophobicity' required for insertion into the ER membrane is very similar in S. cerevisiae and in mammalian cells. Further, when comparing the contributions to the apparent free energy of membrane insertion of the 20 natural amino acids between the S. cerevisiae and the mammalian ER, we find that the two scales are strongly correlated but that the absolute difference between the most hydrophobic and most hydrophilic residues is approximately 2-fold smaller in S. cerevisiae.
"However, when shorter sequence segments were used as input (24–35 residues with charged residues located at the centre), TOPCONS predicted single-pass helices (results not shown). For all studied segments, the ΔG app values are in the range as the translocon integrated TM peptides (−0.60 to 2.54 kcal/mol) (Table S1)   . "
[Show abstract][Hide abstract] ABSTRACT: Charged and polar amino acids in the transmembrane domains of integral membrane proteins can be crucial for protein function and also promote helix-helix association or protein oligomerization. Yet, our current understanding is still limited on how these hydrophilic amino acids are efficiently translocated from the Sec61/SecY translocon into the cell membrane during the biogenesis of membrane proteins. In hepatitis C virus, the putative transmembrane segments of envelope glycoproteins E1 and E2 were suggested to heterodimerize via a Lys-Asp ion-pair in the host endoplasmic reticulum. Therefore in this work, we carried out molecular dynamic simulations in explicit lipid bilayer and solvent environment to explore the stability of all possible bridging ion-pairs using the model of H-segment helix dimers. We observed that, frequently, several water molecules penetrated from the interface into the membrane core to stabilize the charged and polar pairs. The hydration time and amount of water molecules in the membrane core depended on the position of the charged residues as well as on the type of ion-pairs. Similar microsolvation events were observed in simulations of the putative E1-E2 transmembrane helix dimers. Simulations of helix monomers from other members of the Flaviviridae family suggest that these systems show similar behaviors. Thus this study illustrates the important contribution of water microsolvation to overcome the unfavorable energetic cost of burying charged and polar amino acids in membrane lipid bilayers. Also, it emphasizes the novel role of bridging charged or polar interactions stabilized by water molecules in the hydrophobic lipid bilayer core that has an important biological function for helix dimerization in several envelope glycoproteins from the family of Flaviviridae viruses.
"Interestingly, the values, although generally similar, were different for different systems (mammalian ER in vitro and in vivo, yeast, bacteria, and biophysical measurements ). From our measurements in yeast, we obtained ⌬G Leu app ϭ Ϫ0.51 kcal/mol, ⌬G Ala app ϭ 0.12 kcal/mol and an interpolated 3.6 leucines in a 19-residues oligo-alanine sequence for 50% integration (compared with previous measurements in yeast of Ϫ0.21 kcal/mol, 0.06 kcal/mol, and 4.4 leucines, respectively (Hessa et al., 2009). An alternative model to partitioning is a kinetically controlled mechanism in which H-segments trigger the opening of the lateral gate for (irreversible) exit into the lipid phase. "
[Show abstract][Hide abstract] ABSTRACT: The Sec61 translocon mediates the translocation of proteins across the endoplasmic reticulum membrane and the lateral integration of transmembrane segments into the lipid bilayer. The structure of the idle translocon is closed by a lumenal plug domain and a hydrophobic constriction ring. To test the function of the apolar constriction, we have mutated all six ring residues of yeast Sec61p to more hydrophilic, bulky, or even charged amino acids (alanines, glycines, serines, tryptophans, lysines, or aspartates). The translocon was found to be surprisingly tolerant even to the charge mutations in the constriction ring, because growth and translocation efficiency were not drastically affected. Most interestingly, ring mutants were found to affect the integration of hydrophobic sequences into the lipid bilayer, indicating that the translocon does not simply catalyze the partitioning of potential transmembrane segments between an aqueous environment and the lipid bilayer but that it also plays an active role in setting the hydrophobicity threshold for membrane integration.
Molecular biology of the cell 03/2010; 21(10):1662-70. DOI:10.1091/mbc.E10-01-0060 · 4.47 Impact Factor
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