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 ΔF508 CFTR Pool in the Endoplasmic Reticulum Is Increased by Calnexin Overexpression

2004 ◽  
Vol 15 (2) ◽  
pp. 563-574 ◽  
Author(s):  
Tsukasa Okiyoneda ◽  
Kazutsune Harada ◽  
Motohiro Takeya ◽  
Kaori Yamahira ◽  
Ikuo Wada ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Endoplasmic Reticulum ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Cystic Fibrosis Transmembrane ◽  
Erad Pathway

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, ΔF508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of ΔF508 CFTR with calnexin, an ER chaperone, results in the ERAD of ΔF508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of ΔF508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the ΔF508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated ΔF508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of ΔF508 CFTR in the ER.

scholarly journals Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum–associated degradation

2011 ◽  
Vol 22 (16) ◽  
pp. 2797-2809 ◽  
Author(s):  
Yoshihiro Matsumura ◽  
Larry L. David ◽  
William R. Skach
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Proteasome System ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Free System ◽  
Highly Sensitive ◽  
Ubiquitin Proteasome ◽  
Cystic Fibrosis Transmembrane ◽  
Competing Pathways

The Hsp/c70 cytosolic chaperone system facilitates competing pathways of protein folding and degradation. Here we use a reconstituted cell-free system to investigate the mechanism and extent to which Hsc70 contributes to these co- and posttranslational decisions for the membrane protein cystic fibrosis transmembrane conductance regulator (CFTR). Hsc70 binding to CFTR was destabilized by the C-terminal domain of Bag-1 (CBag), which stimulates client release by accelerating ADP-ATP exchange. Addition of CBag during CFTR translation slightly increased susceptibility of the newly synthesized protein to degradation, consistent with a profolding function for Hsc70. In contrast, posttranslational destabilization of Hsc70 binding nearly completely blocked CFTR ubiquitination, dislocation from the endoplasmic reticulum, and proteasome-mediated cleavage. This effect required molar excess of CBag relative to Hsc70 and was completely reversed by the CBag-binding subdomain of Hsc70. These results demonstrate that the profolding role of Hsc70 during cotranslational CFTR folding is counterbalanced by a dominant and essential role in posttranslational targeting to the ubiquitin-proteasome system. Moreover, the degradative outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is in turn governed by the integrated expression of regulatory cochaperones.

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.

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 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.

CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases

1996 ◽  
Vol 271 (2) ◽  
pp. C650-C657 ◽  
Author(s):  
W. W. Reenstra ◽  
K. Yurko-Mauro ◽  
A. Dam ◽  
S. Raman ◽  
S. Shorten
Keyword(s):  
Chloride Channel ◽  
Kinase Inhibitor ◽  
Maximal Activity ◽  
Calyculin A ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Nih 3T3 ◽  
Cystic Fibrosis Transmembrane

We have previously shown [B. Illek, H. Fischer, G. F. Santos, J. H. Widdicombe, T. E. Machen, and W. W. Reenstra, Am. J. Physiol. 268 (Cell Physiol. 37): C886-C893, 1995] that genistein, a tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in NIH/3T3 cells that have been stably transfected with an expression vector for the CFTR (NIH-CFTR cells). In this study, we present evidence suggesting that both genistein and the serine/threonine protein phosphatase (PPase) inhibitor calyculin A activate the CFTR by inhibiting PPase activity. As measured by 125I efflux, genistein and calyculin A stimulate the CFTR to approximately 50% of the maximal activity with forskolin. Neither agonist increases CFTR activity at saturating forskolin concentrations, but genistein and calyculin A have an additive effect on CFTR activity. Forskolin, but neither genistein nor calyculin A, stimulates protein kinase A(PKA) activity. The PKA inhibitor H-89 inhibits CFTR activation and in vivo phosphorylation by all three agonists. Proteolytic digestion of in vivo phosphorylated CFTR suggests that the CFTR is phosphorylated on the same sites during stimulation with genistein and forskolin but on different sites stimulation with calyculin A. The data suggest that genistein and calyculin A inhibit different PPase activities, allowing CFTR phosphorylation and partial stimulation, by a basal PKA activity.

Actin filament organization is required for proper cAMP-dependent activation of CFTR

1999 ◽  
Vol 277 (6) ◽  
pp. C1160-C1169 ◽  
Author(s):  
Adriana G. Prat ◽  
C. Casey Cunningham ◽  
G. Robert Jackson ◽  
Steven C. Borkan ◽  
Yihan Wang ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Single Channel ◽  
Fold Increase ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Whole Cell ◽  
Excised Patches ◽  
Cystic Fibrosis Transmembrane ◽  
Molecular Nature

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(−)]. cAMP stimulation of ABP(−) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl− currents. In ABP(−) cells expressing CFTR, cAMP was also without effect on Cl− conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl− currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(−)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl−channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl− channel activity in excised patches of ABP(−)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

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