Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

Geest, Marleen van; Lolkema, Juke S.

doi:10.1128/mmbr.64.1.13-33.2000

Cited by 178 publications

(179 citation statements)

References 213 publications

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“…The PE-dependent topological organization of PheP was shown through differences in accessibility by membrane-impermeable sulfhydryl reagents to single Cys replacements in the extramembrane domains connecting TMs. The advantage of this technique is that modification is restricted to a single amino acid residue that is less likely to disturb the overall protein structure (29). This idea was also supported by the maintenance of PheP function in cells with natural phospholipid composition.…”

Section: Fig 4 Determination Of Phep Topology In Pe-containing (A)mentioning

confidence: 52%

Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

Zhang

Bogdanov

et al. 2003

Journal of Biological Chemistry

103

122

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Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.Although considerable progress has been made in understanding the assembly of multispanning-membrane proteins (1, 2), the precise molecular events involved in the insertion, orientation, and proper formation of tertiary and quaternary structures of proteins in the membrane are not well defined. Most investigations have been focused on the role of amino acid sequence in directing the assembly of membrane proteins, whereas only a limited number of reports have addressed the effects of the native lipid environment in determining the correct insertion, folding, and topology of membrane proteins. Therefore, there is currently little understanding of, or ability to predict, how membrane protein topogenesis occurs in a given lipid environment. Whether there are constraints imposed on the topological organization of membrane proteins by phospholipid composition in addition to simply providing an amphipathic environment for maintenance of membrane protein conformation is also not clear.The most compelling evidence for a specific role for lipids in membrane protein topological organization is the requirement for phosphatidylethanolamine (PE) 1 for the proper orientation of the 12 transmembrane domains (TMs) of the lactose permease LacY of Escherichia coli (3). Assembly in the absence of PE results in a topological inversion of the N-terminal six TMs and their associated extramembrane domains. PE is required in a late step of maturation for the proper folding of the periplasmic extramembrane domain (P7) linking TMs VII and VIII (4). Proper folding of this domain is required for active (but not facilitated) transport of LacY substrates (5). The native topological org...

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Section: Fig 4 Determination Of Phep Topology In Pe-containing (A)mentioning

confidence: 52%

Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

Zhang

Bogdanov

et al. 2003

Journal of Biological Chemistry

103

122

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“…2B) (37). In contrast, whereas the yeast ortholog (PEM2) is proposed to be similarly polytopic, one portion of the yeast protein contains a hydrophobic stretch of 31 amino acids, which, intriguingly, is the minimum length re- Endoproteinase Lys-C cleavage sites are denoted by arrows, and the length of cleavage fragments generated from proteolysis at each site, as measured from the N-terminal HA-tagged epitope, are indicated in kDa.…”

Section: Discussionmentioning

confidence: 99%

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Shields¹,

Lehner²,

Agellon³

et al. 2003

Journal of Biological Chemistry

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In liver, phosphatidylethanolamine is converted to phosphatidylcholine through a series of three sequential methylation reactions. Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes each transmethylation reaction, and S-adenosylmethionine is the methyl group donor. Biochemical analysis of human liver revealed that the methyltransferase activity is primarily localized to the endoplasmic reticulum and mitochondria-associated membranes. Bioinformatic analysis of the predicted amino acid sequence suggested that the enzyme adopts a polytopic conformation in those membranes. To elucidate the precise membrane topography of PEMT and thereby provide the basis for in-depth functional characterization of the enzyme, we performed endoproteinase-protection analysis of epitopetagged, recombinant protein. Our data suggest a topographical model of PEMT in which four transmembrane regions span the membrane such that both the N and C termini of the enzyme are localized external to the ER. Two hydrophilic connecting loops protrude into the luminal space of the microsomes whereas a corresponding loop on the cytosolic side remains proximate to the membrane. Further support for this model was obtained following endoproteinase-protection analysis of mutant recombinant PEMT derivatives in which specific protease cleavage sites had been genetically engineered or ablated.

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“…We employed an experimental system that has been widely applied to the topology analysis of plasma membrane proteins (Kast et al, 1998;van Geest et al, 2000), but here applied uniquely to a membrane protein on an intracellular organelle. Insertion of a small HA-tag was expected to have the least influence on the cellular behavior of OA1-GFP.…”

Section: Discussionmentioning

confidence: 99%

The ocular albinism type 1 gene product, OA1, spans intracellular membranes 7 times

Sone

Orlow

2007

Experimental Eye Research

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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 Cterminus 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.

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Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

Cited by 178 publications

References 213 publications

Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

The ocular albinism type 1 gene product, OA1, spans intracellular membranes 7 times

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