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.

7 Reads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Scientific Reports 10/2013; 3:3019. DOI:10.1038/srep03019 · 5.58 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    PLoS ONE 09/2012; 7(9):e44263. DOI:10.1371/journal.pone.0044263 · 3.23 Impact Factor
  • Source
    • "It obeys the “positive inside” rule in which there is a preponderance of positively charged residues on the cytoplasmic side of the membrane-spanning sequence[20]. Most type II proteins contain an internal signal-anchor sequence that serves both to initiate translocation and to anchor the polypeptide chain in the lipid bilayer[21], [22]. A few type II proteins have a uncleaved, N-terminal signal sequence, but unlike the case of Ctm-PrP, this sequence serves as a membrane anchor[23]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrP(C)) to the pathogenic one (PrP(Sc)). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and caspase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed. The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis.
    PLoS ONE 01/2011; 6(1):e14602. DOI:10.1371/journal.pone.0014602 · 3.23 Impact Factor
Show more

Similar Publications


7 Reads
Available from