Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae.
ABSTRACT 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.
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ABSTRACT: Membranes are hydrophobic barriers that define the outer boundaries and internal compartments of living cells. Membrane proteins are the gates in these barriers, and they perform vital functions in the highly regulated transport of matter and information across membranes. Membrane proteins destined for the endoplasmic reticulum are targeted either co- or post-translationally to the Sec61 translocon, the major translocation machinery in eukaryotic cells, which allows for lateral partitioning of hydrophobic segments into the lipid bilayer. This thesis aims to acquire insights into the mechanism of membrane protein insertion and the role of different translocon components in targeting, insertion and topogenesis, using the yeast Saccharomyces cerevisiae as a model organism. By measuring the insertion efficiency of a set of model proteins, we studied the sequence requirements for Sec61-mediated insertion of an α-helical transmembrane segment and established a ‘biological hydrophobicity scale’ in yeast, which describes the individual contributions of the 20 amino acids to insertion. Systematic mutagenesis and photo-crosslinking of the Sec61 translocon revealed key residues in the lateral gate that modulate the threshold hydrophobicity for membrane insertion and transmembrane segment orientation. Further, my studies demonstrate that the translocon-associated Sec62 is important not only for post-translational targeting, but also for the insertion and topogenesis of moderately hydrophobic signal anchor proteins and the C-terminal translocation of multi-spanning membrane proteins. Finally, nuclearly encoded mitochondrial membrane proteins were found to evade mis-targeting to the endoplasmic reticulum by containing short C-terminal tails.12/2013, Degree: PhD, Supervisor: Gunnar von Heijne
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ABSTRACT: The Sec61 translocon forms a pore to translocate polypeptide sequences across the membrane and offers a lateral gate for membrane integration of hydrophobic (H) segments. A central constriction of six apolar residues has been shown to form a seal, but also to determine the hydrophobicity threshold for membrane integration: Mutation of these residues in yeast Sec61p to glycines, serines, aspartates, or lysines lowered the hydrophobicity required for integration; mutation to alanines increased it. Whereas four leucines distributed in an oligo-alanine H segment were sufficient for 50% integration, we now find four leucines in the N-terminal half of the H segment to produce significantly more integration than in the C-terminal half, suggesting functional asymmetry within the translocon. Scanning a cluster of three leucines through an oligo-alanine H segment showed high integration levels, except around the position matching that of the hydrophobic constriction in the pore where integration was strongly reduced. Both asymmetry and the position effect of H-segment integration disappeared upon mutation of the constriction residues to glycines or serines, demonstrating that hydrophobicity at this position within the translocon is responsible for the phenomenon. Asymmetry was largely retained, however, when constriction residues were replaced by alanines. These results reflect on the integration mechanism of transmembrane domains and show that membrane insertion of H segments strongly depends not only on their intrinsic hydrophobicity but also on the local conditions in the translocon interior. Thus, the contribution of hydrophobic residues in the H segment is not simply additive and displays cooperativeness depending on their relative position.Proceedings of the National Academy of Sciences 11/2013; · 9.81 Impact Factor
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ABSTRACT: This review discusses main features of transmembrane (TM) proteins which distinguish them from water-soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co-translational and post-translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large-scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen-bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp residues correspond to the outer part of the lipid acyl chain region - between double bonds and carbonyl groups of lipids. These "midpolar" regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side-chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein-lipid binding.Protein Science 06/2014; · 2.86 Impact Factor