Protein 4.2 interaction with hereditary spherocytosis mutants of the cytoplasmic domain of human anion exchanger 1

2010 ◽  
Vol 433 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Susan P. Bustos ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Plasma Membrane ◽  
Anion Exchanger ◽  
Hereditary Spherocytosis ◽  
Cytoplasmic Domain ◽  
Membrane Localization ◽  
Wild Type ◽  
Anion Exchanger 1 ◽  
Plasma Membrane Localization ◽  
293 Cells ◽  
Protein 4.2

AE1 (anion exchanger 1) and protein 4.2 associate in a protein complex bridging the erythrocyte membrane and cytoskeleton; disruption of the complex results in unstable erythrocytes and HS (hereditary spherocytosis). Three HS mutations (E40K, G130R and P327R) in cdAE1 (the cytoplasmic domain of AE1) occur with deficiencies of protein 4.2. The interaction of wild-type AE1, AE1HS mutants, mdEA1 (the membrane domain of AE1), kAE1 (the kidney isoform of AE1) and AE1SAO (Southeast Asian ovalocytosis AE1) with protein 4.2 was examined in transfected HEK (human embryonic kidney)-293 cells. The HS mutants had wild-type expression levels and plasma-membrane localization. Protein 4.2 expression was not dependent on AE1. Protein 4.2 was localized throughout the cytoplasm and co-localized at the plasma membrane with the HS mutants mdAE1 and kAE1, but at the ER (endoplasmic reticulum) with AE1SAO. Pull-down assays revealed diminished levels of protein 4.2 associated with the HS mutants relative to AE1. The mdAE1 did not bind protein 4.2, whereas kAE1 and AE1SAO bound wild-type amounts of protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had decreased plasma-membrane localization compared with wild-type protein 4.2, and co-expression with AE1 enhanced its plasma-membrane localization. Subcellular fractionation showed the majority of wild-type and G2A/C173A protein 4.2 was associated with the cytoskeleton of HEK-293 cells. The present study shows that cytoplasmic HS mutants cause impaired binding of protein 4.2 to AE1, leaving protein 4.2 susceptible to loss during erythrocyte development.

Effect of Cytosolic Hereditary Spherocytosis Mutations of Human Erythrocyte Anion Exchanger 1 on Its Interaction with Cytoskeletal Protein 4.2.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3742-3742
Author(s):  
Susan P. Bustos ◽  
Reinhart A.F. Reithmeier
Keyword(s):  
Anion Exchanger ◽  
Fusion Proteins ◽  
Structural Changes ◽  
Hereditary Spherocytosis ◽  
Cytoskeletal Protein ◽  
Red Cell ◽  
Wild Type ◽  
Anion Exchanger 1 ◽  
E Coli ◽  
Protein 4.2

Abstract Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Human AE1 has two functionally independent domains: its 52 kDa C-terminal membrane domain catalyzes the exchange of chloride for bicarbonate across the membrane while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the membrane to the cytoskeleton, giving the red cell its stability and flexibility. Several proteins bind to cdb3 including cytoskeletal protein 4.2, ankyrin, glycolytic enzymes and deoxyhemoglobin. Three mutations in cdb3 (E40K, G130R and P327R) are associated with the hemolytic anemia hereditary spherocytosis (HS) and decreased levels of erythrocyte protein 4.2 while maintaining a normal amount of AE1 at the red cell membrane. Wild-type and mutant cdb3 proteins were expressed in E. coli and purified and it was shown through a variety of biophysical methods that these three HS mutations do not cause major structural changes in this domain. Each of these mutations introduces a positive charge at the surface of the protein that is predominantly negatively charged, which may have an effect on protein interactions while maintaining its native folded structure. Full-length wild-type AE1 or HS mutants were co-expressed with protein 4.2 in HEK-293 cells in order to study their interaction in a mammalian cell line. All three HS mutant proteins were expressed at similar levels to wild-type in these cells and were shown to be present at the membrane using immunofluorescence and confocal microscopy. These proteins were also co-expressed in LLCPK-1 cells for the purpose of co-localization studies using immunofluorescence. A series of GST fusion proteins of protein 4.2 domains were designed based on a homology model of protein 4.2 and regions of the protein known to interact with AE1. These fusion proteins were expressed in E. coli and purified on glutathione-Sepharose resin and were used to study their interaction with purified wild-type and HS mutant cdb3 proteins in vitro. Blot overlay analysis showed that a protein 4.2 GST fusion protein containing a putative β-hairpin region (Asp145 to Glu203) binds specifically to wild-type cdb3 while GST does not. It is hypothesized that since these three HS mutations do not cause major structural changes in cdb3, the decreased level of protein 4.2 in the red cells of these patients is a result of impaired binding that occurs due to these mutation sites.

scholarly journals Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Jolene Ramsey ◽  
Emily C. Renzi ◽  
Randy J. Arnold ◽  
Jonathan C. Trinidad ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Wild Type Virus ◽  
Membrane Localization ◽  
Clear Understanding ◽  
Wild Type ◽  
Plasma Membrane Localization ◽  
C Terminus ◽  
N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

scholarly journals The carboxyl-terminally truncated kidney anion exchanger 1 R901X dRTA mutant is unstable at the plasma membrane

2016 ◽  
Vol 310 (9) ◽  
pp. C764-C772 ◽  
Author(s):  
Ensaf Almomani ◽  
Rawad Lashhab ◽  
R. Todd Alexander ◽  
Emmanuelle Cordat
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Anion Exchanger ◽  
Basolateral Membrane ◽  
Recycling Rate ◽  
Wild Type ◽  
Anion Exchanger 1 ◽  
Renal Epithelial Cells ◽  
Gene Coding ◽  
Renal Tubular

Mutations in the SLC4A1 gene coding for kidney anion exchanger 1 (kAE1) cause distal renal tubular acidosis (dRTA). We investigated the fate of the most common truncated dominant dRTA mutant kAE1 R901X. In renal epithelial cells, we found that kAE1 R901X is less abundant than kAE1 wild-type (WT) at the plasma membrane. Although kAE1 WT and kAE1 R901X have similar half-lives, the decreased abundance of kAE1 R901X at the surface is due to an increased endocytosis rate and a decreased recycling rate of endocytosed proteins. We propose that, in polarized renal epithelial cells, the apically mistargeted kAE1 R901X mutant is endocytosed faster than kAE1 WT and its recycling to the basolateral membrane is delayed. This resets the equilibrium, such that kAE1 R901X resides predominantly in an endomembrane compartment, thereby likely participating in development of dRTA disease.

scholarly journals Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface

2004 ◽  
Vol 378 (3) ◽  
pp. 1015-1021 ◽  
Author(s):  
Joanne C. CHEUNG ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Palmitic Acid ◽  
Anion Exchanger ◽  
Cell Surface Expression ◽  
Co2 Removal ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.

Dual functional significance of calcineurin homologous protein 1 binding to Na+/H+ exchanger isoform 1

2011 ◽  
Vol 301 (2) ◽  
pp. C280-C288 ◽  
Author(s):  
Masafumi Matsushita ◽  
Hiroo Tanaka ◽  
Keiji Mitsui ◽  
Hiroshi Kanazawa
Keyword(s):  
Plasma Membrane ◽  
Binding Site ◽  
Functional Significance ◽  
Gene Knockout ◽  
Membrane Localization ◽  
Wild Type ◽  
Homologous Protein ◽  
Plasma Membrane Localization ◽  
Dual Functional ◽  
Associated Proteins

Calcineurin homologous protein 1 (CHP1) binds to the hydrophilic tail of the Na+/H+ exchanger isoform 1 (NHE1). Previous gene knockout of CHP1 revealed that the loss of CHP1 caused a decrease in the total amount of NHE1, suggesting the destabilization of NHE1 molecules without CHP1 (Matsushita et al., Am J Physiol Cell Physiol 293: C246–C254, 2007). However, Pang et al. ( J Biol Chem 276: 17367–17372, 2001) reported that NHE1 without a CHP1 binding site was found in the plasma membrane, suggesting no requirement of CHP1 binding for plasma membrane localization of NHE1. Here, the functional significance of CHP1 binding to NHE1 was examined to resolve these contradictory results. In CV1 cells, which overexpressed wild-type NHE1, overexpression of CHP1 caused an increase in both the total amount of NHE1 and the colocalization of NHE1 and CHP1 at the plasma membrane. This provided new visual evidence of the localization of NHE1 from endoplasmic reticulum to the plasma membrane upon CHP1 binding. An immunoprecipitation assay showed that the expression of CHP1 reduced the ubiquitination of NHE1 and/or its associated proteins. Mutant NHE1s without CHP1 binding site exhibited a modest localization to the plasma membrane. After reaching the plasma membrane, these mutant NHE1s exhibited shorter half-lives than the wild-type NHE1 with CHP1. The results suggest a dual functional significance of CHP1 and its binding region: 1) binding of CHP1 stabilizes NHE1 and increases its plasma membrane localization by masking a NHE1 disposal signal, and 2) CHP1 binding is required for the antiporter activity.

scholarly journals Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA

2021 ◽  
pp. 100607
Author(s):  
Ivana Malcova ◽  
Ladislav Bumba ◽  
Filip Uljanic ◽  
Darya Kuzmenko ◽  
Jana Nedomova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Binding ◽  
Membrane Localization ◽  
Effector Protein ◽  
Plasma Membrane Localization
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