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

1998 ◽  
Vol 76 (5) ◽  
pp. 729-733 ◽  
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.

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

1996 ◽  
Vol 318 (2) ◽  
pp. 645-648 ◽  
Author(s):  
Lisa Y TAM ◽  
Carolina LANDOLT-MARTICORENA ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Band 3 ◽  
Site Directed Mutagenesis ◽  
Acceptor Site ◽  
Oligosaccharyl Transferase ◽  
Transmembrane Segments ◽  
Free Translation ◽  
Reticulocyte Lysates ◽  
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.

scholarly journals Proteolysis of ankyrin and of band 3 protein in chemically induced cell fusion. Ca2+ is not mandatory for fusion

1984 ◽  
Vol 218 (2) ◽  
pp. 295-305 ◽  
Author(s):  
R D A Lang ◽  
C Wickenden ◽  
J Wynne ◽  
J A Lucy
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Cysteine Proteinase ◽  
Serine Proteinase ◽  
Band 3 ◽  
Human Erythrocytes ◽  
General Mechanism ◽  
Band 3 Protein ◽  
Fusion Reactions ◽  
Chlorpromazine Hydrochloride

Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide a general mechanism for the fusion of lipid bilayers in biomembrane fusion reactions.

scholarly journals Transmembrane domain–dependent partitioning of membrane proteins within the endoplasmic reticulum

2008 ◽  
Vol 181 (1) ◽  
pp. 105-118 ◽  
Author(s):  
Paolo Ronchi ◽  
Sara Colombo ◽  
Maura Francolini ◽  
Nica Borgese
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Fluorescent Proteins ◽  
Lipid Interactions ◽  
Er Exit Sites ◽  
Physicochemical Features ◽  
Er Membrane

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein–protein and protein–lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail–anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER–Golgi boundary by simple receptor-independent mechanisms based on partitioning.

scholarly journals Dissection of De Novo Membrane Insertion Activities of Internal Transmembrane Segments of ATP-Binding-Cassette Transporters: Toward Understanding Topological Rules for Membrane Assembly of Polytopic Membrane Proteins

1998 ◽  
Vol 9 (4) ◽  
pp. 853-863 ◽  
Author(s):  
Jian-Ting Zhang ◽  
Mingang Chen ◽  
Ernest Han ◽  
Changsen Wang
Keyword(s):  
Membrane Proteins ◽  
De Novo ◽  
Chinese Hamster ◽  
Membrane Insertion ◽  
Strong Activity ◽  
Membrane Assembly ◽  
Transmembrane Segments ◽  
Model Protein ◽  
Tm Domain ◽  
Polytopic Membrane Protein

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an Nin-Cout rather than the predicted Nout-Cin orientation, and TM3 is in cytoplasm rather than the predicted Nin-Coutorientation in the membrane. It is possible that TM4 has a strong activity to initiate the Nin-Cout membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo Nin-Cout membrane insertion activity whereas TM4 initiates the Nin-Cout membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates Nin-Cout membrane insertion whereas TM4 stops the insertion event; and 2) “leaving one TM segment out of the membrane” may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the Nin-Cout membrane insertion activities of the TM segments.

scholarly journals Membrane proteins in human erythrocytes during cell fusion induced by oleoylglycerol

1978 ◽  
Vol 176 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Susan J. Quirk ◽  
Quet Fah Ahkong ◽  
Gaynor M. Botham ◽  
Jan Vos ◽  
Jack A. Lucy
Keyword(s):  
Membrane Proteins ◽  
Cell Fusion ◽  
Gel Electrophoresis ◽  
Band 3 ◽  
Human Erythrocytes ◽  
Proteolytic Degradation ◽  
Fracture Face ◽  
Band 3 Protein ◽  
Osmotic Lysis ◽  
Very High

1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca2+ used, at least 80% of the cells fused after 30min at 37°C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in ‘ghosts’ prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca2+, although some cells fused when no exogenous Ca2+ was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of ‘ghosts’ prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1–2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca2+ into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed.

scholarly journals Asparagine-linked oligosaccharides are localized to single extracytosolic segments in multi-span membrane glycoproteins

1994 ◽  
Vol 302 (1) ◽  
pp. 253-260 ◽  
Author(s):  
C Landolt-Marticorena ◽  
R A F Reithmeier
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Na Channel ◽  
Acceptor Site ◽  
Membrane Glycoproteins ◽  
Transmembrane Segments ◽  
Comprehensive Survey ◽  
Single Polypeptide ◽  
Average Size

A comprehensive survey of mammalian multi-span (polytopic) membrane proteins showed that asparagine(N)-linked oligosaccharides are localized to single extracytosolic segments. In most membrane proteins this is because potential consensus sites for N-glycosylation (Asn-Xaa-Ser/Thr, X not equal to Pro) are not found in multiple extracytosolic segments. In functional proteins where consensus N-glycosylation sites are contained within more than one extracytosolic segment, only the first segment contains N-linked carbohydrate. An exception is the alpha-subunit of the Na+ channel, which consists of a duplicated structure containing two glycosylated segments. The average size of established N-glycosylated loops connecting two transmembrane segments is 62 residues, with the smallest glycosylated loop being 33 residues in size. N-glycosylated sites are more highly conserved than non-glycosylated (primarily cytosolic) sites and are more common toward the N-terminus of the membrane domain of multi-span membrane proteins. The optimal conditions for glycosylation of consensus sites within an extracytosolic domain of a multi-span membrane protein are (i) the acceptor site is well-spaced (greater than 10 residues) from the transmembrane domain, (ii) the loop is greater than 30 residues in size and (iii) the segment is the first in the protein to contain a suitable extracytosolic consensus site. The localization of N-linked oligosaccharide chains to a single protein segment suggests either glycosylation of multiple loops may compromise protein folding or function, or only a single polypeptide domain can be optimally glycosylated during biosynthesis in vivo.

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