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: 4.6). 04/1997; 272(10):6119-27. DOI: 10.1074/jbc.272.10.6119
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Our previous study showed that the Autographa californica nucleopolyhedrovirus (AcMNPV) ac76 gene is essential for both budded virion (BV) and occlusion-derived virion (ODV) development. More importantly, deletion of ac76 affects intranuclear microvesicle formation. However, the exact role by which ac76 affects virion morphogenesis remains unknown. In this report, we characterized the expression, distribution, and topology of Ac76 to further understand the functional role of Ac76 in virion morphogenesis. Ac76 contains an α-helical transmembrane domain, and phase separation showed that it was an integral membrane protein. In AcMNPV-infected cells, Ac76 was detected as a stable dimer that was resistant to SDS and thermal denaturation, and only a trace amount of monomer was detected. A co-immunoprecipitation assay demonstrated the dimerization of Ac76 by high affinity self-association. Western blot analyses of purified virions and their nucleocapsid and envelope fractions showed that Ac76 was associated with the envelope fractions of both BV and ODV. Immunoelectron microscopy revealed that Ac76 was localized to the plasma membrane, endoplasmic reticulum (ER), nuclear membrane, intranuclear microvesicles, and ODV envelope. Amino acids 15-48 of Ac76 were identified as an atypical inner nuclear membrane-sorting motif because it was sufficient to target fusion proteins to the ER and nuclear membrane in the absence of viral infection and to the intranuclear microvesicles and ODV envelope during infection. Topology analysis of Ac76 by selective permeabilization showed that Ac76 was a type II integral membrane protein with an N-terminus exposed to the cytosol and a C-terminus hidden in the ER lumen.
    Journal of Virology 11/2013; · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra- and inter-cellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative α-helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments insert into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components.
    Journal of Virology 12/2013; · 4.65 Impact Factor
  • Source
    [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. · 5.08 Impact Factor