scholarly journals Complementation studies with co-expressed fragments of human red cell band 3 (AE1): the assembly of the anion-transport domain in Xenopus oocytes and a cell-free translation system

1998 ◽  
Vol 332 (1) ◽  
pp. 161-171 ◽  
Author(s):  
Jonathan D. GROVES ◽  
Lin WANG ◽  
Michael J. A. TANNER
Keyword(s):  
Xenopus Oocytes ◽  
Band 3 ◽  
Translation System ◽  
Red Cell ◽  
Membrane Domain ◽  
Cytoplasmic Loop ◽  
Free System ◽  
Anion Transporter ◽  
Free Translation ◽  
The Individual

We examined the assembly of the membrane domain of the human red cell anion transporter (band 3; AE1) by co-expression of recombinant N- and C-terminal fragments in Xenopus oocytes and in cell-free translation with canine pancreatic microsomes. Co-immunoprecipitation was performed in non-denaturing detergent solutions using antibodies directed against the N- and C-termini of the membrane domain. Eleven of the twelve fragments were expressed stably in oocytes in the presence or absence of their respective partners. However, the fragment containing from putative span nine to the C-terminus could be detected in oocytes only when co-expressed with its complementary partner containing the first eight spans. Co-expression of pairs of fragments divided in the first, second, third and fourth exofacial loops and in the fourth cytoplasmic loop resulted in a concentration-dependent association, but a pair of fragments divided in the sixth cytoplasmic loop did not co-immunoprecipitate. When two complementary fragments were translated separately in the cell-free system and the purified microsomes were then mixed, co-immunoprecipitation was observed only if the membranes were first fused using polyethylene glycol. This shows that co-immunoprecipitation results from specific interactions within the membrane and is not an artefact of detergent solubilization or immunoprecipitation. We demonstrate that band 3 assembly can occur within the membrane after translation, insertion and initial folding of the individual fragments have been completed. We conclude that most band 3 fragments contain the necessary information to fold in the membrane and adopt a structure that is sufficiently similar to the native protein that it permits correct assembly with its complementary partner.

scholarly journals Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1)

1999 ◽  
Vol 344 (3) ◽  
pp. 699-711 ◽  
Author(s):  
Jonathan D. GROVES ◽  
Michael J. A. TANNER
Keyword(s):  
Structural Model ◽  
Functional Expression ◽  
Band 3 ◽  
Current Evidence ◽  
Translation System ◽  
Cdna Clones ◽  
Red Cell ◽  
Loss Of Function ◽  
Free Translation ◽  
High Level

We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.

Hereditary Stomatocytosis Associated with a Loss of Function Mutation In Rh-Associated Glycoprotein (RhAG)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2040-2040
Author(s):  
Connie M Westhoff ◽  
Seth Alper
Keyword(s):  
Xenopus Oocytes ◽  
Collecting Duct ◽  
Monovalent Cation ◽  
Band 3 ◽  
Cell Permeability ◽  
Red Cell ◽  
Loss Of Function ◽  
Wild Type ◽  
Water Control ◽  
Glycophorin B

Abstract Abstract 2040 The erythroid Rh family of proteins includes RhCE and RhD which carry the common Rh antigens, and the related Rh-associated glycoprotein, RhAG. RhAG is required for trafficking of the blood group proteins to the membrane and forms the core of a macro-complex in the membrane which includes glycophorin B, Band 3, CD47, and LW. The Rh proteins are structurally and functionally related to the Amt superfamily of NH3/NH4+ transport proteins, and RhAG and its nonerythroid paralogs, RhCG and RhBG, have been shown to mediate NH3/NH4+ transport. RhCG is responsible for part of renal collecting duct epithelial cell NH3/NH4+ secretion, and Rhcg-/- mice exhibit incomplete distal renal tubular acidosis due to impaired urinary NH4+ excretion. The Rhag-/- mouse is grossly normal, and the significance of RhAG-mediated NH3/NH4+ transport in human erythrocytes remains unclear. Over-hydrated hereditary stomatocytosis (OHSt) is a rare dominant disorder characterized by moderate hemolytic anemia, increased mean red cell volumes, stomatocytes and echinocytes, and increased red cell permeability to the monovalent cations, Na+ and K+. Six of the seven OHSt kindred studied by Bruce et al. (Blood. 2009;113:1350) displayed a heterozygous Phe65Ser mutation in RhAG. Expression studies of the mutant 65Ser-RhAG in Xenopus oocytes induced a monovalent cation flux compatible with the cation leak seen in RBCs. The increased Na+ and decreased K+ contents of mutant RhAG-expressing oocytes suggested that F65S is a gain-of-function mutation that opens a cation leak, likely within the RhAG polypeptide. In this study the ammonia transport properties of the OHSt mutant 65Ser-RhAG were investigated. Xenopus oocytes were injected with cRNA encoding wild-type RhAG, the OHSt mutant 65Ser-RhAG, and 65Val-RhAG, an engineered mutation with a smaller hydrophobic side chain at position 65. Wild-type and mutant RhAG polypeptides were well-expressed in the oocyte membrane as measured by quantitative immunoblotting. Uptake of the NH3/NH4+ substrate analog 14C-methylammonium (MA), was assayed in oocytes previously injected with water (control) or with cRNA. Expression of wild-type RhAG mediated MA uptake at rates 6-fold greater than that of water-injected controls. Uptake of MA by oocytes expressing 65Val-RhAG was equivalent to that of wild type RhAG. However, MA uptake by oocytes expressing OHSt mutant 65Ser-RhAG was greatly reduced to less than 20% that of oocytes expressing wild-type RHAG or 65Val-RhAG, and was only 1.5-fold greater than that of water-injected control oocytes. Co-expression with other, individual Rh complex members glycophorin B, RhD, RhCE, or Band 3 did not alter MA-mediated uptake by RhAG-expressing oocytes. Importantly, this study reveals that the RhAG mutation Phe65Ser found in patients with type 1 over-hydrated stomatocytosis is a loss of function mutation. Further study is required to define the relationship between loss of NH3/NH4+ transport and erythrocyte Na+ and K+ cation content. Disclosures: Westhoff: Immucor: Scientific Advisor.

scholarly journals Development of a cell-free system to study the membrane assembly of photosynthetic proteins of Rhodobacter capsulatus.

1990 ◽  
Vol 111 (1) ◽  
pp. 87-94 ◽  
Author(s):  
D Troschel ◽  
M Müller
Keyword(s):  
Rhodobacter Capsulatus ◽  
Photosynthetic Apparatus ◽  
Membrane Vesicles ◽  
Translation System ◽  
Intracytoplasmic Membrane ◽  
Free System ◽  
Light Harvesting Complex ◽  
Free Translation ◽  
A Cell

A cell-free translation system from the facultatively photoheterotrophic bacterium Rhodobacter capsulatus is described. Synthesis of two proteins of the bacterium's photosynthetic apparatus (light-harvesting complex B870 alpha and beta) was performed by SP6 polymerase transcription of the subcloned genes, isolation of the mRNA and translation in vitro using a cell-free extract of R. capsulatus cells. The integration of these proteins in vitro into added intracytoplasmic membrane vesicles (ICM) is demonstrated. Without addition of ICM approximately 70% of the synthesized B870 proteins were soluble. If, however, ICM were present during synthesis, the majority of the soluble protein was found to associate with the membranes. The membrane-associated polypeptides could be solubilized only by detergent treatment but could not be extracted by treatment at alkaline pH (Na2CO3), suggesting that the proteins had been firmly inserted into the lipid bilayer. Moreover, the B870 alpha and beta proteins that integrated in vitro into ICM were also found to associate with pigment ligands and to assemble into a native reaction center/B870 complex. The native conformation of this complex isolated from ICM by Triton fractionation was demonstrated by microspectral analysis of the bound pigments.

scholarly journals Topology studies with biosynthetic fragments identify interacting transmembrane regions of the human red-cell anion exchanger (band 3; AE1)

1999 ◽  
Vol 344 (3) ◽  
pp. 687-697 ◽  
Author(s):  
Jonathan D. GROVES ◽  
Michael J. A. TANNER
Keyword(s):  
Anion Exchanger ◽  
Band 3 ◽  
Glycosylation Site ◽  
Intramolecular Interactions ◽  
Red Cell ◽  
Chloride Uptake ◽  
C Terminus ◽  
Free Translation ◽  
Membrane Spanning ◽  
General Method

The red-cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and is N-glycosylated at Asn-642. We have shown that the integrity of six different loops are not essential for stilbene disulphonate-sensitive chloride uptake in Xenopus oocytes. We used an N-glycosylation mutagenesis approach to examine the orientation of the N-terminus and the endogenous glycosylation site of each C-terminal fragment by cell-free translation. The fragments initiating in the loops preceding spans 2, 9 and 11 did not insert into the membrane with the expected orientation. Furthermore, N-glycosylation of Asn-642 might facilitate the membrane integration of span 7. The correct integration of spans 2-3 required the presence of the region containing span 4 and that the luminal exposure of the C-terminus of span 7 is increased in the presence of the region including span 6 or span 8. The results suggest the span 8 region is required for the correct folding of spans 9-10, at least in the presence of the span 11-12 region. Our results suggest that there are intramolecular interactions between the regions of transmembrane spans 1 and 2, 2 and 4, 4 and 5, 7 and 8, 8 and 9-10, and 9-10 and 11-12. Spans 1, 4, 5, 6 and 8 might act as a scaffold for the assembly of spans 2-3, 7 and 9-10. This approach might provide a general method for dissecting the interactions between membrane-spanning regions of polytopic membrane proteins.

scholarly journals The expression of the abnormal human red cell anion transporter from South-East Asian ovalocytes (band 3 SAO) inXenopusoocytes

FEBS Letters ◽  
1993 ◽  
Vol 330 (2) ◽  
pp. 186-190 ◽  
Author(s):  
Jonathan D. Groves ◽  
Susan M. Ring ◽  
Ann E. Schofield ◽  
Michael J.A. Tanner
Keyword(s):  
East Asian ◽  
Band 3 ◽  
Red Cell ◽  
Anion Transporter

scholarly journals Complementation Studies with Co-expressed Fragments of the Human Red Cell Anion Transporter (Band 3; AE1)

1997 ◽  
Vol 272 (16) ◽  
pp. 10631-10638 ◽  
Author(s):  
Lin Wang ◽  
Jonathan D. Groves ◽  
William J. Mawby ◽  
Michael J. A. Tanner
Keyword(s):  
Band 3 ◽  
Red Cell ◽  
Anion Transporter

NMR Solution Structure of a Cytoplasmic Surface Loop of the Human Red Cell Anion Transporter, Band 3†

Biochemistry ◽  
1998 ◽  
Vol 37 (33) ◽  
pp. 11670-11678 ◽  
Author(s):  
David Askin ◽  
Graham B. Bloomberg ◽  
Eric J. Chambers ◽  
Michael J. A. Tanner
Keyword(s):  
Solution Structure ◽  
Band 3 ◽  
Red Cell ◽  
Nmr Solution Structure ◽  
Surface Loop ◽  
Cytoplasmic Surface ◽  
Anion Transporter
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