Retargeting of the mitochondrial protein p32/gC1Qr to a cytoplasmic compartment and the cell surface

p32/gC1qR is a small acidic protein that has been reported to have a broad range of distinct functions and to associate with a wide array of cellular, viral and bacterial proteins. It has been found in each of the main cellular compartments including mitochondria, nucleus and cytoplasm and is also thought to be located at the plasma membrane and secreted into the extracellular matrix. The true physiological role(s) of p32 remains controversial because it has been difficult to reconcile all of the findings on protein interactions and the seemingly disparate observations on compartmentalisation. However, it has been proposed that p32 is somehow involved in transport processes connecting diverse cellular compartments and the cell surface. Here we show that native p32 appears to be localised mainly in the mitochondria and is not detectable on the cell surface. However, addition of a short tag to the N-terminus of p32 appears to block its mitochondrial targeting, resulting in redirection into a cytoplasmic vesicular pattern, overlapping with the endoplasmic reticulum. The redirection of p32 results in an alteration in and co-localisation with ER markers including calreticulin, a lumenal ER chaperone. Furthermore, we show both by immunofluorescence and cross-linking studies that this also results in cell-surface expression of p32. These results indicate that, at least under certain circumstances, p32 can be retargeted and may help to provide an explanation for the diverse observations on its localization.

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Position 97 of HLA-B, a residue implicated in pathogenesis of ankylosing spondylitis, plays a key role in cell surface free heavy chain expression

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2016-209512 ◽

2016 ◽

Vol 76 (3) ◽

pp. 593-601 ◽

Cited By ~ 9

Author(s):

Liye Chen ◽

Hui Shi ◽

Jack Yuan ◽

Paul Bowness

Keyword(s):

Ankylosing Spondylitis ◽

Cell Surface ◽

Heavy Chain ◽

Protein Interactions ◽

Hela Cells ◽

Antigen Processing ◽

Surface Expression ◽

Cell Surface Expression ◽

Expression Levels ◽

Free Heavy Chain

ObjectiveAssociation of position 97 (P97) residue polymorphisms in human leucocyte antigen (HLA)-B, including HLA-B*27, with ankylosing spondylitis (AS) has recently been reported. We studied the effect of P97 variations on cell surface expression of the AS-associated HLA-B*27 and HLA-B*51, and the AS-protective HLA-B*7.MethodsFlow cytometry was used to measure surface expression of HLA-B*27 in C1R/HeLa cells expressing HLA-B*27 (N97) and six mutants at P97 (N97T, N97S, N97V, N97R, N97W and N97D). Transporter associated with antigen processing-deficient T2, tapasin-deficient 220, β2m-deficient HCT15 and endoplasmic reticulum aminopeptidase 1 or β2m-clustered regularly interspaced short palindromic repeats/Cas9-knockout HeLa cells were used to provide evidence for specific protein interactions. Surface expression of HLA-B*7/HLA-B*51 P97 mutants was also studied.ResultsMutation of HLA-B*27 P97 to the AS risk residue threonine increased cell surface free heavy chain (FHC) expression. Protective residues (serine or valine) and non-AS-associated residues (arginine or tryptophan) did not alter FHC expression. The N97D mutation reduced expression of conventional and FHC forms of HLA-B*27. Differences in FHC expression levels between HLA-B*27, HLA-B*27-N97T and HLA-B*27-N97D were dependent on the presence of functional β2m. HLA-B*7, which has an AS-protective serine at P97, expressed lower levels of FHC than HLA-B*27 or HLA-B*51. Introduction of asparagine at P97 of both HLA-B*7 and HLA-B*51 increased FHC expression.ConclusionsThe nature of P97 residue affects surface expression of HLA-B*27, B*7 and B*51, with AS-associated residues giving rise to higher FHC expression levels. The association of P97 amino acid polymorphisms with AS could be, at least in part, explained by its effect on HLA-B*27 FHC cell surface expression.

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Low HLA-C expression at cell surfaces correlates with increased turnover of heavy chain mRNA.

Journal of Experimental Medicine ◽

10.1084/jem.181.6.2085 ◽

1995 ◽

Vol 181 (6) ◽

pp. 2085-2095 ◽

Cited By ~ 124

Author(s):

J A McCutcheon ◽

J Gumperz ◽

K D Smith ◽

C T Lutz ◽

P Parham

Keyword(s):

Cell Surface ◽

Heavy Chain ◽

Protein Interactions ◽

Stop Codon ◽

Mrna Level ◽

Surface Expression ◽

Cell Surface Expression ◽

Protein Protein Interactions ◽

Peptide Binding Groove ◽

Beta 2 Microglobulin

In comparison with HLA-A and -B, the protein products of the HLA-C locus are poorly characterized, in part because of their low level of expression at the cell surface. Here, we examine how protein-protein interactions during assembly and regulation of the mRNA level affect cell surface expression of HLA-C. We find that intrinsic properties of the HLA-C heavy chain proteins do not correlate with low cell surface expression: HLA-C heavy chains associate and dissociate with beta 2-microglobulin (beta 2m) at rates comparable to those found for HLA-A and -B, and increased competition for beta 2m does not alter the surface expression of HLA-C. From studies of chimeric genes spliced from the HLA-B7 and -Cw3 genes, we find that chimeric proteins containing the B7 peptide-binding groove can have low cell surface expression, suggesting that inefficiency in binding peptides is not the cause of low cell surface expression for HLA-C. The surface levels of HLA-A, -B, or -C in cells transfected with cDNA can be similar, implicating noncoding regions of HLA-C heavy chain genes in the regulation of surface expression. We find that HLA-C mRNA is expressed at lower levels than HLA-B mRNA and that this difference results from faster degradation of the HLA-C message. Experiments examining chimeric B7/Cw3 and B7/Cw6 genes suggest that a region determining low expression of HLA-C is to be found between the 3' end of exon 3 and a site in the 3' untranslated region, approximately 600 bases downstream of the translation stop codon.

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High-Content Imaging for Large-Scale Detection of Low-Affinity Extracellular Protein Interactions

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219879053 ◽

2019 ◽

Vol 24 (10) ◽

pp. 987-999

Author(s):

Laura Wood ◽

Gavin J. Wright

Keyword(s):

Cell Surface ◽

Protein Interactions ◽

Large Scale ◽

Local Environment ◽

Surface Expression ◽

Extracellular Protein ◽

Automated Image Analysis ◽

Cell Surface Expression ◽

High Content Imaging ◽

Pathogen Invasion

Extracellular protein interactions coordinate cellular responses with their local environment and have important roles in pathogen invasion and disease. Due to technical challenges associated with studying binding events at the cell surface, the systematic and reliable identification of novel ligand–receptor pairs remains difficult. Here, we describe the development of a cell-based assay using large-scale transient transfections and high-content imaging (HCI) to detect extracellular binding events. We optimized the parameters for efficient transfection of human cells with cDNA plasmids encoding full-length cell surface receptors in 384-well plates. Using a range of well-characterized structurally diverse low-affinity cell surface interactions, we show that transfected cells probed with highly avid ligands can be used to successfully identify ligand–receptor pairs using an HCI platform and automated image analysis software. To establish the high-throughput potential of this approach, we also screened a pool of ligands against a collection of 2455 cell surface expression clones and found that known ligand–receptor interactions could be robustly and consistently detected across the library using this technology.

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The Arabidopsis Abiotic Stress-Induced TSPO-Related Protein Reduces Cell-Surface Expression of the Aquaporin PIP2;7 through Protein-Protein Interactions and Autophagic Degradation

The Plant Cell ◽

10.1105/tpc.114.134080 ◽

2014 ◽

Vol 26 (12) ◽

pp. 4974-4990 ◽

Cited By ~ 75

Author(s):

Charles Hachez ◽

Vasko Veljanovski ◽

Hagen Reinhardt ◽

Damien Guillaumot ◽

Celine Vanhee ◽

...

Keyword(s):

Abiotic Stress ◽

Cell Surface ◽

Protein Interactions ◽

Surface Expression ◽

Cell Surface Expression ◽

Related Protein ◽

Protein Protein Interactions ◽

Autophagic Degradation

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CFTR-associated ligand is a negative regulator of Mrp2 expression

AJP Cell Physiology ◽

10.1152/ajpcell.00100.2016 ◽

2017 ◽

Vol 312 (1) ◽

pp. C40-C46 ◽

Cited By ~ 1

Author(s):

Man Li ◽

Carol J. Soroka ◽

Kathy Harry ◽

James L. Boyer

Keyword(s):

Cell Surface ◽

Protein Interactions ◽

Posttranscriptional Regulation ◽

Pdz Domain ◽

Rat Hepatocytes ◽

Surface Expression ◽

Negative Regulator ◽

Cell Surface Expression ◽

Binding Motif ◽

Sodium Hydrogen Exchanger

The multidrug resistance-associated protein 2 (Mrp2) is an ATP-binding cassette transporter that transports a wide variety of organic anions across the apical membrane of epithelial cells. The expression of Mrp2 on the plasma membrane is regulated by protein-protein interactions. Cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) interacts with transmembrane proteins via its PDZ domain and reduces their cell surface expression by increasing lysosomal degradation and intracellular retention. Our results showed that CAL is localized at the trans-Golgi network of rat hepatocytes. The expression of CAL is increased, and Mrp2 expression is decreased, in the liver of mice deficient in sodium/hydrogen exchanger regulatory factor-1. To determine whether CAL interacts with Mrp2 and is involved in the posttranscriptional regulation of Mrp2, we used glutathione S-transferase (GST) fusion proteins with or without the COOH-terminal PDZ binding motif of Mrp2 as the bait in GST pull-down assays. We demonstrated that Mrp2 binds to CAL via its COOH-terminal PDZ-binding motif in GST pull-down assays, an interaction verified by coimmunoprecipitation of these two proteins in cotransfected COS-7 cells. In COS-7 and LLC-PK1 cells transfected with Mrp2 alone, only a mature, high-molecular-mass band of Mrp2 was detected. However, when cells were cotransfected with Mrp2 and CAL, Mrp2 was expressed as both mature and immature forms. Biotinylation and streptavidin pull-down assays confirmed that CAL dramatically reduces the expression level of total and cell surface Mrp2 in Huh-7 cells. Our findings suggest that CAL interacts with Mrp2 and is a negative regulator of Mrp2 expression.

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Actinin-1 binds to the C-terminus of A2B adenosine receptor (A2BAR) and enhances A2BAR cell-surface expression

Biochemical Journal ◽

10.1042/bcj20160272 ◽

2016 ◽

Vol 473 (14) ◽

pp. 2179-2186 ◽

Cited By ~ 7

Author(s):

Ying Sun ◽

Wenbao Hu ◽

Xiaojie Yu ◽

Zhengzhao Liu ◽

Robert Tarran ◽

...

Keyword(s):

Cell Surface ◽

Protein Interactions ◽

Adenosine Receptor ◽

Point Mutations ◽

Surface Expression ◽

Cell Surface Expression ◽

Protein Protein Interactions ◽

Physiological Conditions ◽

A2b Adenosine Receptor ◽

C Terminus

A2BAR (A2B adenosine receptor) has been implicated in several physiological conditions, such as allergic or inflammatory disorders, vasodilation, cell growth and epithelial electrolyte secretion. For mediating the protein–protein interactions of A2BAR, the receptor's C-terminus is recognized to be crucial. In the present study, we unexpectedly found that two point mutations in the A2BAR C-terminus (F297A and R298A) drastically impaired the expression of A2BAR protein by accelerating its degradation. Thus we tested the hypothesis that these two point mutations disrupt A2BAR's interaction with a protein essential for A2BAR stability. Our results show that both mutations disrupted the interaction of A2BAR with actinin-1, an actin-associated protein. Furthermore, actinin-1 binding stabilized the global and cell-surface expression of A2BAR. By contrast, actinin-4, another non-muscle actinin isoform, did not bind to A2BAR. Thus our findings reveal a previously unidentified regulatory mechanism of A2BAR abundance.

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Abstract 468: Identification of a New Component of the Na v 1.5 Complex: αB-crystallin Interacts with Na v 1.5 and Regulates Ubiquitination and Internalization of Cell Surface Na v 1.5

Circulation Research ◽

10.1161/res.119.suppl_1.468 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Yuan Huang ◽

Zhijie Wang ◽

Yinan Liu ◽

Qiuyun Chen ◽

Qing k. Wang

Keyword(s):

Cell Surface ◽

Protein Interactions ◽

Sodium Current ◽

Surface Expression ◽

Expression Level ◽

Cell Surface Expression ◽

Protein Protein Interactions ◽

Interacting Protein ◽

Binding Partner ◽

Αb Crystallin

Mutations in cardiac Na + channel Na v 1.5 cause cardiac arrhythmias and sudden death. The cardiac Na + channel functions as a protein complex, however, its complete components remain to be fully elucidated. A yeast two-hybrid screen identified a new candidate Na v 1.5-interacting protein, αB-crystallin. GST-pull-down, co-immunoprecipitation and immunostaining analyses validated the interaction between Na v 1.5 and αB-crystallin. Overexpression of αB-crystallin significantly increased peak sodium current ( I Na ) density, and the underlying molecular mechanism is the increased cell surface expression level of Na v 1.5 via reduced internalization of cell surface Na v 1.5 and ubiquitination of Na v 1.5. Knockout of αB-crystallin expression significantly decreased the cell-surface expression level of Na v 1.5. αB-crystallin interacted with Nedd4-2, however, a catalytically inactive Nedd4-2-C801S mutant reduced the interaction between αB-crystallin and Nedd4-2 and also blocked the increase of peak I Na density by αB-crystallin. Na v 1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of αB-crystallin on reduction of Na v 1.5 ubiquitination and increases of I Na density. The data suggest that the interactions between αB-crystallin and functionally active Nedd4-2 and between αB-crystallin and Na v 1.5 are critical to increased I Na density by αB-crystallin. Multiple mutations in αB-crystallin have been associated with human diseases. Two mutations, R109H and R151X, eliminated the effect of αB-crystallin on I Na . This study identifies αB-crystallin as a new binding partner for Na v 1.5. αB-crystallin interacts with Na v 1.5 and increases I Na by modulating the expression level and internalization of cell surface Na v 1.5 and ubiquitination of Na v 1.5, which requires the protein-protein interactions between αB-crystallin and Na v 1.5 and between αB-crystallin and functionally active Nedd4-2.

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