scholarly journals Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells

2001 ◽  
Vol 281 (3) ◽  
pp. C908-C921 ◽  
Author(s):  
Grischa Chandy ◽  
Michael Grabe ◽  
Hsiao-Ping H. Moore ◽  
Terry E. Machen
Keyword(s):  
Fluorescent Protein ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelia ◽  
Ratiometric Imaging ◽  
Green Fluorescent ◽  
Respiratory Epithelial ◽  
Cystic Fibrosis Transmembrane

Work addressing whether cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pHG) of HeLa cells was 6.4, while pHG of mutant (ΔF508) and wild-type CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7–7.0. Comparison of genetically matched ΔF508 and WT-CFTR cells showed that the absence of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H+ vacuolar (V)-ATPase countered by a H+leak, which was unaffected by CFTR. To estimate Golgi proton permeability ( P H+ ), we modeled transient changes in pHG induced by inhibiting the V-ATPase and by acidifying the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi P H+ = 7.5 × 10−4 cm/s when pHG = 6.5, and surprisingly, P H+ decreased as pHG decreased.

Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice

1992 ◽  
Vol 2 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Jeffrey A. Whitsett ◽  
Chitta R. Dey ◽  
Barry R. Stripp ◽  
Kathryn A. Wikenheiser ◽  
Jean C. Clark ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial ◽  
Cystic Fibrosis Transmembrane

scholarly journals Control of Cellular GADD34 Levels by the 26S Proteasome

2008 ◽  
Vol 28 (23) ◽  
pp. 6989-7000 ◽  
Author(s):  
Matthew H. Brush ◽  
Shirish Shenolikar
Keyword(s):  
Fluorescent Protein ◽  
26S Proteasome ◽  
Initiation Factor ◽  
Transmembrane Conductance Regulator ◽  
Heterologous Proteins ◽  
Transmembrane Conductance ◽  
Physiological Importance ◽  
Response To Stress ◽  
Green Fluorescent ◽  
Cystic Fibrosis Transmembrane

ABSTRACT GADD34, the product of a growth arrest and DNA damage-inducible gene, is expressed at low levels in unstressed cells. In response to stress, the cellular content of GADD34 protein increases and, on termination of stress, rapidly declines. We investigated the mechanisms that control GADD34 levels in human cells. GADD34 proteins containing either an internal FLAG or a C-terminal green fluorescent protein epitope were degraded at rates similar to endogenous GADD34. However, the addition of epitopes at the N terminus or deletion of N-terminal sequences stabilized GADD34. N-terminal peptides of GADD34, either alone or fused to heterologous proteins, exhibited rapid degradation similar to wild-type GADD34, thereby identifying an N-terminal degron. Deletion of internal PEST repeats had no impact on GADD34 stability but modulated the binding and activity of protein phosphatase 1. Proteasomal but not lysosomal inhibitors enhanced GADD34 stability and eukaryotic initiation factor 2α (eIF-2α) dephosphorylation, a finding consistent with GADD34's role in assembling an eIF-2α phosphatase. GADD34 was polyubiquitinated, and this modification enhanced its turnover in cells. A stabilized form of GADD34 promoted the accumulation and aggregation of the mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508), highlighting the physiological importance of GADD34 turnover in protein processing in the endoplasmic reticulum and the potential impact of prolonged GADD34 expression in human disease.

scholarly journals COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments

2013 ◽  
Vol 24 (5) ◽  
pp. 633-642 ◽  
Author(s):  
Shogo Kakoi ◽  
Tomohiro Yorimitsu ◽  
Ken Sato
Keyword(s):  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Growth Defects ◽  
Copii Vesicle ◽  
Ubiquitin Proteasome ◽  
Green Fluorescent ◽  
Cystic Fibrosis Transmembrane

Proteins that fail to fold in the endoplasmic reticulum (ER) are subjected to ER-associated degradation (ERAD). Certain transmembrane ERAD substrates are segregated into specialized ER subdomains, termed ER-associated compartments (ERACs), before targeting to ubiquitin–proteasome degradation. The traffic-independent function of several proteins involved in COPII-mediated ER-to-Golgi transport have been implicated in the segregation of exogenously expressed human cystic fibrosis transmembrane conductance regulator (CFTR) into ERACs in Saccharomyces cerevisiae. Here we focus on the properties of COPII components in the sequestration of enhanced green fluorescent protein (EGFP)–CFTR into ERACs. It has been demonstrated that the temperature-sensitive growth defects in many COPII mutants can be suppressed by overexpressing other genes involved in COPII vesicle formation. However, we show that these suppression abilities are not always correlated with the ability to rescue the ERAC formation defect, suggesting that COPII-mediated EGFP-CFTR entry into ERACs is independent of its ER-to-Golgi trafficking function. In addition to COPII machinery, we find that ER-associated Hsp40s are also involved in the sequestration process by directly interacting with EGFP-CFTR. COPII components and ER-associated Hsp40, Hlj1p, act in the same pathway to sequester EGFP-CFTR into ERACs. Our findings point to an as-yet-undefined role of COPII proteins in the formation of ERACs.

scholarly journals Impact of Heterogeneity within Cultured Cells on Bacterial Invasion: Analysis of Pseudomonas aeruginosa andSalmonella enterica Serovar Typhi Entry into MDCK cells by Using a Green Fluorescent Protein-Labelled Cystic Fibrosis Transmembrane Conductance Regulator Receptor

2000 ◽  
Vol 68 (2) ◽  
pp. 861-870 ◽  
Author(s):  
A. Alev Gerçeker ◽  
Tanweer Zaidi ◽  
Peter Marks ◽  
David E. Golan ◽  
Gerald B. Pier
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bacterial Uptake ◽  
Cftr Protein ◽  
Green Fluorescent

ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel that also serves as a receptor for entry ofPseudomonas aeruginosa and Salmonella entericaserovar Typhi into epithelial cells. To evaluate heterogeneity in CFTR protein expression in cultured cells and the effect of heterogeneity on internalization of different P. aeruginosa and serovar Typhi strains, we used two-color flow cytometry and confocal laser microscopy to study bacterial uptake by Madin-Darby canine kidney (MDCK) type I epithelial cells stably expressing a green fluorescent protein (GFP)-CFTR fusion construct (MDCK–GFP-CFTR cells). We found a strong correlation between cell size and GFP-CFTR protein expression, with 60 to 70% of cells expressing low levels of GFP-CFTR protein, 20 to 30% expressing intermediate levels, and <10% expressing high levels. The cells were sorted into low-, intermediate-, or high-level producers of CFTR protein; in vitro growth of each sorted population yielded the same distribution of CFTR protein expression as that in the original population. Cells expressing either low or high levels of CFTR protein internalized bacteria poorly; maximal bacterial uptake occurred in the cells expressing intermediate levels of CFTR protein. Treatment of MDCK cells with sodium butyrate markedly enhanced the production of CFTR protein without increasing cell size; butyrate treatment also increased the proportion of cells with internalized bacteria. However, there were fewer bacteria per butyrate-treated cell and, for P. aeruginosa, there was an overall decrease in the total level of bacterial uptake. The most highly ingested bacterial strains were internalized by fewer total MDCK–GFP-CFTR cells, indicating preferential bacterial uptake by a minority of epithelial cells within a given culture. Confocal fluorescence microscopy showed that P. aeruginosa and serovar Typhi induced cytoplasmic accumulation of CFTR protein close to the plasma membrane where the bacteria were adherent. These results show that within a population of MDCK–GFP-CFTR cells, there are cells with markedly different abilities to ingest bacteria via CFTR, the majority of the P. aeruginosa and serovar Typhi cells are ingested by the one-fourth to one-third of the cells that exhibit an intermediate size and level of CFTR protein expression, and overexpression of the CFTR receptor does not increase total bacterial uptake but rather allows more epithelial cells to ingest fewer total bacteria.

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