Topological Rules for Membrane Protein Assembly in Eukaryotic Cells
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.
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ABSTRACT: The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) has been proposed to be a transmembrane protein; however, its features remain unclear. Here, we used biochemical approaches to determine its topological properties. Our data reveal that TGBp2 is mainly associated with the endoplasmic reticulum membrane. The resistance of TGBp2 in proteoliposomes, prepared from both the BaMV-infected tissues and in vitro reconstitution system, to both chemical extraction and trypsin digestion confirmed that it is indeed an integral membrane protein. On the basis of the minor change in the size of the major stable TGBp2-derived tryptic fragment from the monomeric TGBp2, as well as the sensitivity of the cysteine residues at the C-terminal tail of TGBp2 to maleimide modification, we suggest that TGBp2 adopts a topology with both its short N- and C-terminal tails exposed to the outer surface of the endoplasmic reticulum. Moreover, TGBp2 is able to self-assemble as revealed by the significant increase in multimeric TGBp2 when the TGBp2-containing proteoliposomes were treated with chemical crosslinker or oxidation agent.Virology 09/2008; 379(1):1-9. DOI:10.1016/j.virol.2008.06.019 · 3.28 Impact Factor
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ABSTRACT: OA1 (GPR143) is a pigment cell-specific intracellular glycoprotein consisting of 404 amino acid residues that is mutated in patients with ocular albinism type 1, the most common form of ocular albinism. While its cellular localization is suggested to be endolysosomal and melanosomal, the physiological function of OA1 is currently unclear. Recent reports predicted that OA1 functions as a G protein coupled receptor (GPCR) based on its weak amino acid sequence similarity to known GPCRs, and on demonstration of GPCR activity in OA1 mislocalized to the plasma membrane. Because mislocalization of proteins is often caused by or induces defects in their proper folding/assembly, the significance of these studies remains unclear. A characteristic feature of GPCRs is a seven transmembrane domain structure. We analyzed the membrane topology of OA1 properly localized to intracellular lysosomal organelles in COS-1 cells. To accomplish this analysis, we established experimental conditions that allowed selective permeabilization of the plasma membrane while leaving endolysosomal membranes intact. Domains were mapped by the insertion of a hemagglutinin (HA) tag into the predicted cytosolic/luminal regions of OA1 molecule and the accessibility of tag to HA antibody was determined by immunofluorescence. HA-tagged lysosome associated membrane protein 1 (LAMP1), a type I membrane protein, was employed as a reporter for selective permeabilization of the plasma membrane. Our results show experimentally that the C-terminus of OA1 is directed to the cytoplasm and that the protein spans the intracellular membrane 7 times. Thus, OA1, properly localized intracellularly, is a 7 transmembrane domain integral membrane protein consistent with its putative role as an intracellular GPCR.Experimental Eye Research 01/2008; 85(6):806-16. DOI:10.1016/j.exer.2007.08.016 · 3.02 Impact Factor
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ABSTRACT: The nuclear pore complex is the predominant structure in the nuclear envelope that spans the double nuclear membranes of all eukaryotes. Yeasts have one additional organelle that is also embedded in the nuclear envelope: the spindle pole body, which functions as the microtubule organizing center. The only protein known to localize to and be important in the assembly of both of these yeast structures is the integral membrane protein, Ndc1p. However, no homologues of Ndc1p had been characterized in metazoa. Here, we identify and analyze NDC1 homologues that are conserved throughout evolution. We show that the overall topology of these homologues is conserved. Each contains six transmembrane segments in its N-terminal half and has a large soluble C-terminal half of approximately 300 amino acids. Charge distribution analysis infers that the N- and C-termini are exposed to the cytoplasm. Limited proteolysis of yeast Ndc1p in cellular membranes confirms the orientation of its C-terminus. Although it is not known whether vertebrate NDC1 protein localizes to nuclear pores like its yeast counterpart, the human homologue contains three FG repeats in the C-terminus, a feature of many nuclear pore proteins. Moreover, a small region containing mutations that affect assembly of the nuclear pore in yeast is highly conserved throughout evolution. Lastly, we bring together data from another study to demonstrate that the human homologue of NDC1 is the known inner nuclear membrane protein, NET3.The Anatomical Record Part A Discoveries in Molecular Cellular and Evolutionary Biology 07/2006; 288(7):681-94. DOI:10.1002/ar.a.20335