Topological Rules for Membrane Protein Assembly in Eukaryotic Cells

Department of Biochemistry, Stockholm University, S-106 91 Stockholm, Sweden.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/1997; 272(10):6119-27. DOI: 10.1074/jbc.272.10.6119
Source: PubMed


Insertion into the endoplasmic reticulum membrane of model proteins with one, two, and four transmembrane segments and different distributions of positively charged residues in the N-terminal tail and the polar loops has been studied both in vitro and in vivo. Membrane insertion of these same constructs has previously been analyzed in Escherichia coli, thus making possible a detailed comparison between the topological rules for membrane protein assembly in prokaryotic and eukaryotic cells. In general, we find that positively charged residues have similar effects on the membrane topology in both systems when they are placed in the N-terminal tail but that the effects of charged residues in internal loops clearly differ. Our results rule out a sequential start-stop transfer model where successive hydrophobic segments insert with alternating orientations starting from the most N-terminal one as the only mechanism for membrane protein insertion in eukaryotic cells.

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    • "Therefore, we concluded that the P4H enzyme responsible for post-translational rhEPO modification is located in the secretory compartments, i.e. the endoplasmic reticulum (ER) or the Golgi apparatus. All sequences display a hydrophobic segment near the N-terminus following a positively charged residue, suggesting a localization of the proteins in the secretory compartments40. However, we examined the subcellular localization of the seven moss P4Hs in silico with four different programs based on different algorithms. "
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    ABSTRACT: Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.
    Full-text · Article · Oct 2013 · Scientific Reports
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    • "The insertion of proteins into membranes is thought to be achieved by a variety of conserved translocases and integrases (such as the well-described Sec translocon) acting both independently and cooperatively (Samuelson et al. 2000; Dalbey et al. 2011; Nishiyama et al. 2012). The addition of positive charges to the N-termini of transmembrane proteins can prevent the translocation of the termini across membranes in both E. coli and eukaryotes (Gafvelin et al. 1997), whether they require the main Sec protein-conducting channel (Li et al. 1988; Yamane and Mizushima 1988) or not (Whitley et al. 1994). Although the prevalence of the positive-inside rule is recognized , the mechanisms by which positive charges exert their topogenic effects are not well understood. "
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    ABSTRACT: In the great majority of genomes, the use of positive charge increases, on average, approaching protein N-termini. Such charged residues slow ribosomes by interacting with the negatively charged exit tunnel. This has been proposed to be selectively advantageous as it provides an elongation speed ramp at translational starts. Positive charges, however, are known to orientate proteins in membranes by the positive-inside rule whereby excess charge lies on the cytoplasmic side of the membrane. Which of these two models better explains the N-terminal loading of positively charged amino acids? We find strong evidence that the tendency for average positive charge use to increase at termini is exclusively due to membrane protein topology: 1) increasing N-terminal positive charge is not found in cytosolic proteins, but in transmembrane ones with cytosolic N-termini, with signal sequences contributing additional charge; 2) positive charge density at N-termini corresponds to the length of cytoplasmically exposed transmembrane tails, its usage increasing just up until the membrane; 3) membrane-related patterns are repeated at C-termini, where no ramp is expected; and 4) N-terminal positive charge patterns are no different from those seen internally in proteins in membrane-associated domains. The overall apparent increase in positive charge across all N-termini results from membrane proteins using positive charge adjacent to the cytosolic leaflet, combined with a skewed distribution of where N-termini cross the plasma membrane; 5) while Escherichia coli was predicted to have a 5' ribosomal occupancy ramp of at least 31 codons, in contrast to what is seen in yeast, we find in ribosomal footprinting data no evidence for such a ramp. In sum, we find no need to invoke a translational ramp to explain the rising positive charge densities at N-termini. The membrane orientation model makes a full account of the trend.
    Preview · Article · Sep 2013 · Molecular Biology and Evolution
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    • "To assess the effect of the presence of ionizable residues on the GpA TM segment insertion into biological membranes, we located this hydrophobic sequence (Fig. 4A) in place of the second TM fragment of the well-characterized Escherichia coli inner membrane protein leader peptidase (Lep). Although of bacterial origin, Lep integrates efficiently into dog pancreas microsomes with the same topology as in E. coli [30] (i.e., with both the N- and C-termini exposed to the luminal side of the ER membrane) and the presence of its first TM segment together with the cytoplasmic P1 domain (Figure 4B) is sufficient for proper targeting of chimeric proteins to the eukaryotic membrane [30], [31]. An engineered glycosylation site placed at the C-terminal P2 domain is glycosylated efficiently upon correct insertion into the microsomal membrane (Fig. 4B), serving as a reporter to distinguish between a lumenal (glycosylated) and a cytoplasmic (unglycosylated) location. "
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    ABSTRACT: The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.
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