Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein

N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253–260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.

Download Full-text

A new concept in polytopic membrane proteins following from the study of band 3 protein

Biochemistry and Cell Biology ◽

10.1139/o98-085 ◽

1998 ◽

Vol 76 (5) ◽

pp. 729-733 ◽

Cited By ~ 5

Author(s):

Naotaka Hamasaki ◽

Hiroyuki Kuma ◽

Kazuhisa Ota ◽

Masao Sakaguchi ◽

Katsuyoshi Mihara

Keyword(s):

Membrane Proteins ◽

Transmembrane Domain ◽

Band 3 ◽

Band 3 Protein ◽

Transmembrane Peptides ◽

Lipid Interactions ◽

Transmembrane Segments ◽

Novel Concept ◽

Polytopic Membrane Protein ◽

Peptide Classification

In the present communication, we introduce a novel concept in multispanning polytopic membrane proteins revealed by the study of the band 3 protein. The transmembrane domain of such proteins can be divided into three categories, that is, hydrophilic loops connecting transmembrane peptides (category 1), portions embedded by peptide-peptide interactions (category 2), and portions embedded by peptide-lipid interactions (category 3). Category 2 peptides of polytopic membrane proteins were found to stably reside in the lipid bilayer without peptide-lipid interactions that had been thought to be essential for transmembrane segments. Category 3 peptides are equivalent to single-spanning segments of bitopic membrane proteins. Three different experiments, namely proteolytic digestion, chemical modification of the band 3 protein, and cell free transcription and translation, were used to categorize the transmembrane peptides.Key words: band 3 protein, transmembrane (TM) peptide, classification of TM, category 2-TM, polytopic membrane protein.

Download Full-text

Transmembrane folding of the human erythrocyte anion exchanger (AE1, Band 3) determined by scanning and insertional N-glycosylation mutagenesis

Biochemical Journal ◽

10.1042/bj3390269 ◽

1999 ◽

Vol 339 (2) ◽

pp. 269-279 ◽

Cited By ~ 28

Author(s):

Milka POPOV ◽

Jing LI ◽

Reinhart A. F. REITHMEIER

Keyword(s):

Anion Exchanger ◽

Human Erythrocyte ◽

Band 3 ◽

Translation System ◽

Acceptor Site ◽

Oligosaccharide Structure ◽

Hek 293 ◽

Transmembrane Segments ◽

Free Translation ◽

A Cell

The human erythrocyte anion exchanger (AE1, Band 3) contains up to 14 transmembrane segments, with a single site of N-glycosylation at Asn642 in extracellular (EC) loop 4. Scanning and insertional N-glycosylation mutagenesis were used to determine the folding pattern of AE1 in the membrane. Full-length AE1, when expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells, retained a high-mannose oligosaccharide structure. Scanning N-glycosylation mutagenesis of EC loop 4 showed that N-glycosylation acceptor sites (Asn-Xaa-Ser/Thr) spaced 12 residues from the ends of adjacent transmembrane segments could be N-glycosylated. An acceptor site introduced at position 743 in intracellular (IC) loop 5 that could be N-glycosylated in a cell-free translation system was not N-glycosylated in transfected cells. Mutations designed to disrupt the folding of this loop enhanced the level of N-glycosylation at Asn743in vitro. The results suggest that this loop might be transiently exposed to the lumen of the endoplasmic reticulum during biosynthesis but normally folds rapidly, precluding N-glycosylation. EC loop 4 insertions into positions 428, 484, 754 and 854 in EC loops 1, 2, 6 and 7 respectively were efficiently N-glycosylated, showing that these regions were extracellular. EC loop 4 insertions into positions 731 or 785 were poorly N-glycosylated, which was inconsistent with an extracellular disposition for these regions of AE1. Insertion of EC loop 4 into positions 599 and 820 in IC loops 3 and 6 respectively were not N-glycosylated in cells, which was consistent with a cytosolic disposition for these loops. Inhibitor-affinity chromatography with 4-acetamido-4´-isothiocyanostilbene-2,2´-disulphonate (SITS)-Affi-Gel was used to assess whether the AE1 mutants were in a native state. Mutants with insertions at positions 428, 484, 599, 731 and 785 showed impaired inhibitor binding, whereas insertions at positions 754, 820 and 854 retained binding. The results indicate that the folding of the C-terminal region of AE1 is more complex than originally proposed and that this region of the transporter might have a dynamic aspect.

Download Full-text

Sequence requirements for membrane assembly of polytopic membrane proteins: molecular dissection of the membrane insertion process and topogenesis of the human MDR3 P-glycoprotein.

Molecular Biology of the Cell ◽

10.1091/mbc.7.11.1709 ◽

1996 ◽

Vol 7 (11) ◽

pp. 1709-1721 ◽

Cited By ~ 22

Author(s):

J T Zhang

Keyword(s):

Amino Acid ◽

Membrane Proteins ◽

Amino Acid Sequences ◽

Membrane Insertion ◽

Terminal Amino Acid ◽

Membrane Assembly ◽

P Glycoprotein ◽

Free Translation ◽

Terminal Amino ◽

Polytopic Membrane Protein

The biogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the biogenesis and membrane assembly of membrane proteins with multiple TM segments is still incomplete because of the complexity and diversity of polytopic membrane proteins. In an attempt to investigate further the biogenesis of polytopic membrane proteins, I used the human MDR3 P-glycoprotein (Pgp) as a model polytopic membrane protein and expressed it in a coupled cell-free translation/translocation system. I showed that the topogenesis of the C-terminal half MDR3 Pgp molecule is different from that of the N-terminal half. This observation is similar to that of the human MDR1 Pgp. The membrane insertion properties of the TM1 and TM2 in the N-terminal half molecule are different. The proper membrane anchorage of both TM1 and TM2 of the MDR3 Pgp is affected by their C-terminal amino acid sequences, whereas only the membrane insertion of the TM1 is dependent on the N-terminal amino acid sequences. The efficient membrane insertion of TM3 and TM5 of MDR3 Pgp, on the other hand, requires the presence of the putative TM4 and TM6, respectively. The TM8 in the C-terminal half does not contain an efficient stop-transfer activity. These observations suggest that the membrane insertion of putative TM segments in the human MDR3 Pgp does not simply follow the prevailing sequential event of the membrane insertion by signal-anchor and stop-transfer sequences. These results, together with my previous findings, suggest that different isoforms of Pgp can be used in comparison as a model system to understand the molecular mechanism of topogenesis of polytopic membrane proteins.

Download Full-text

An ER translocon for multi-pass membrane protein biogenesis

eLife ◽

10.7554/elife.56889 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 7

Author(s):

Philip T McGilvray ◽

S Andrei Anghel ◽

Arunkumar Sundaram ◽

Frank Zhong ◽

Michael J Trnka ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Glutamate Transporter ◽

Cell Physiology ◽

Transmembrane Segments ◽

Cryo Electron Microscopy ◽

Protein Biogenesis ◽

Exit Tunnel ◽

Ribosome Exit Tunnel ◽

Molecular Framework

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Download Full-text

Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild-type and membrane protein knockout mice

Blood ◽

10.1182/blood-2008-03-146159 ◽

2008 ◽

Vol 112 (9) ◽

pp. 3900-3906 ◽

Cited By ~ 66

Author(s):

M. Estela Campanella ◽

Haiyan Chu ◽

Nancy J. Wandersee ◽

Luanne L. Peters ◽

Narla Mohandas ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Knockout Mice ◽

Protein Band ◽

Band 3 ◽

Glycolytic Enzyme ◽

Erythrocyte Membranes ◽

Multienzyme Complexes ◽

Human Erythrocyte Membranes ◽

Ankyrin Protein

Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking α-spectrin, ankyrin, protein 4.2, protein 4.1, β-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3.

Download Full-text