Analysis of Transmembrane Helix Integration in the Endoplasmic Reticulum in S. cerevisiae

ArticleinJournal of Molecular Biology 386(5):1222-8 · April 2009with33 Reads
Impact Factor: 4.33 · DOI: 10.1016/j.jmb.2009.01.027 · Source: PubMed

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.

    • "By placing each of the 20 natural amino acids in the middle of the Ala/Leu based Hsegment individually, we could further determine the individual contribution of each amino acid to the apparent free energy of membrane insertion (ΔG app ) and establish a biological hydrophobicity scale in yeast (Figure 9). membrane insertion were determined using Ala/Leu based H-segments, with the corresponding amino acid placed in the center (Hessa et al., 2009). Reprinted with permission. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Thesis · Dec 2013
    • "The current working hypothesis is that this is because, first, the high protein content of the ER membrane makes it more polar than a pure lipid bilayer [4], and, secondly, the interior of the Sec61 translocon channel through which the nascent polypeptide is threaded across the membrane is less polar than aqueous buffer [5]. Using similar approaches, biological hydrophobicity scales have also been derived for insertion of transmembrane segments into the ER of the yeast Saccharomyces cerevisiae [6] and the inner membrane of Escherichia coli [7]; they are both qualitatively similar to the original Hessa scale. "
    [Show abstract] [Hide abstract] ABSTRACT: Membrane proteins currently receive a lot of attention, in large part thanks to a steady stream of high-resolution X-ray structures. Although the first few structures showed proteins composed of tightly packed bundles of very hydrophobic more or less straight transmembrane α-helices, we now know that helix-bundle membrane proteins can be both highly flexible and contain transmembrane segments that are neither very hydrophobic nor necessarily helical throughout their lengths. This raises questions regarding how membrane proteins are inserted into the membrane and fold in vivo, and also complicates life for bioinformaticians trying to predict membrane protein topology and structure.
    Preview · Article · Jun 2011 · Biochemical Society Transactions
    • "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) [1,9,10]. We used the I-TASSER webserver for tertiary structure predictions for the putative TM segments of the envelope glycoproteins. "
    [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.
    Full-text · Article · Apr 2011 · Biochimica et Biophysica Acta
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