CFTR Expression and ER-Associated Degradation in Yeast

Zhang, Yimao; Michaelis, Susan; Brodsky, Jeffrey L.

doi:10.1385/1-59259-187-6:257

Cited by 29 publications

(38 citation statements)

References 3 publications

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“…Growth of cells beyond 16 hr (post-induction) will give rise to decreased CFTR expression as shown in Figure 4. This is probably due to turnover of the protein, perhaps due to upregulation of the yeast protein quality control machinery [6][7][8][9]13 . It is therefore advisable to monitor CFTR expression levels after induction with galactose, if possible, as the optimal time to harvest the cells may vary from one laboratory to another.…”

Section: Representative Resultsmentioning

confidence: 99%

“…Like many 'difficult' eukaryotic membrane proteins, expression in a fastgrowing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed [4][5][6][7][8][9] . Proteins involved in the processing of recombinant CFTR in yeast have been described [6][7][8][9] .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting.…”

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confidence: 99%

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Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

O'Ryan

Rimington

Cant

et al. 2012

JoVE

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein [1][2][3] . Like many 'difficult' eukaryotic membrane proteins, expression in a fastgrowing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed [4][5][6][7][8][9] . Proteins involved in the processing of recombinant CFTR in yeast have been described [6][7][8][9] .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR constructcontaining yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008) 10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

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Section: Representative Resultsmentioning

confidence: 99%

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confidence: 99%

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

O'Ryan

Rimington

Cant

et al. 2012

JoVE

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“…Predominantly in the ER Membrane-We previously developed yeast expression systems to define and characterize the ERAD requirements for several clinically relevant mammalian ion channels, including the chloride channel CFTR (29,(32)(33)(34) and the trimeric sodium channel, ENaC (13,14). The yeast system can then be exploited to identify the unique degradation requirements for each substrate by examining the protein fate in cells mutated for specific genes, such as those required for ERAD (35).…”

Section: Ncc Can Be Expressed In the Yeast S Cerevisiae And Residesmentioning

confidence: 99%

The Thiazide-sensitive NaCl Cotransporter Is Targeted for Chaperone-dependent Endoplasmic Reticulum-associated Degradation

Needham

Mikoluk

Dhakarwal

et al. 2011

Journal of Biological Chemistry

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Background:The cation chloride cotransporter NCC is degraded by undefined mechanisms. Results: NCC requires specific conserved machinery for chaperone-dependent recognition, ubiquitination, and proteasomal routing. Conclusion: NCC exhibits distinct ERAD requirements, which correlate with its transmembrane topology and distinguish it from other clients. Significance: These ER quality control components process misfolded conformational intermediates of NCC and other structurally related cotransporters that are vital for human health.

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“…The cDNA sequence encoding the Z variant of ␣-1 protease inhibitor (A1PiZ) (McCracken and Kruse, 1993) was cloned either into vectors containing the inducible GAL1 promoter: pYES2.0 (2 , Amp r , URA3; Invitrogen) and pBM743 (CEN/ARS, Amp r , URA3; Invitrogen), or into a vector with the repressible MET25 promoter (Mumberg et al, 1994): p426MET25 (2 , Amp r , URA3; ATCC, Manassas, VA). Triple-hemagglutinin (HA)-tagged cystic fibrosis transmembrane conductance regulator (CFTR) expression in yeast was driven by the constitutive phosphoglycerate kinase promoter in a 2 plasmid (Zhang et al, 2002). The vector p425CyTerm (CEN/ARS, Amp r , LEU2) for constitutive protein expression (see below) was described previously (Palmer et al, 2003).…”

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confidence: 99%

Characterization of anERADGene asVPS30/ATG6Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble Z Variant of Human α-1 Proteinase Inhibitor (A1PiZ) and Another for Aggregates of A1PiZ

Kruse

Brodsky

McCracken

2006

MBoC

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185

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The Z variant of human ␣-1 proteinase inhibitor (A1PiZ) is a substrate for endoplasmic reticulum-associated protein degradation (ERAD). To identify genes required for the degradation of this protein, A1PiZ degradation-deficient (add) yeast mutants were isolated. The defect in one of these mutants, add3, was complemented by VPS30/ATG6, a gene that encodes a component of two phosphatidylinositol 3-kinase (PtdIns 3-kinase) complexes: complex I is required for autophagy, whereas complex II is required for the carboxypeptidase Y (CPY)-to-vacuole pathway. We found that upon overexpression of A1PiZ, both PtdIns 3-kinase complexes were required for delivery of the excess A1PiZ to the vacuole. When the CPY-to-vacuole pathway was compromised, A1PiZ was secreted; however, disruption of autophagy led to an increase in aggregated A1PiZ rather than secretion. These results suggest that excess soluble A1PiZ transits the secretion pathway to the trans-Golgi network and is selectively targeted to the vacuole via the CPY-to-vacuole sorting pathway, but excess A1PiZ that forms aggregates in the endoplasmic reticulum is targeted to the vacuole via autophagy. These findings illustrate the complex nature of protein quality control in the secretion pathway and reveal multiple sites that recognize and sort both soluble and aggregated forms of aberrant or misfolded proteins. INTRODUCTIONCell function and survival depend on protein quality control to identify and remove aberrant proteins. Although two cellular sites of proteolysis are known, the lysosome/vacuole and cytoplasmic 26S proteasome, the recognition of aberrant proteins and mechanisms for delivery to these sites are still being defined. Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control process in which aberrant or misassembled proteins in the secretory pathway are identified and removed (reviewed in Hampton, 2002;Tsai et al., 2002;Kostova and Wolf, 2003;McCracken and Brodsky, 2003). After entering the endoplasmic reticulum (ER), a nascent protein that fails to fold or assemble properly can be "recognized" by the ER quality control machinery, retained within the ER, and then retrotranslocated to the cytoplasm where it is degraded by the proteasome.The recognition of ERAD substrates must exhibit flexibility to distinguish slowly folding proteins from aberrant proteins. Molecular chaperones play a critical role in this selection process; for example, the ER heat-shock protein (Hsp70), BiP, is vital for the selection of soluble substrates and is believed to hold the substrates in an unfolded state competent for retrotranslocation (Nishikawa et al., 2001;Kabani et al., 2003).The complexity of the ERAD pathway has emerged with the identification of new ERAD substrates and the components required for their selection and degradation in yeast. For example, the analysis of topologically distinct ERAD substrates indicates that molecular chaperones in the cytoplasm and in the ER lumen distinguish membrane and soluble substrates, respectively (Huyer et al., 20...

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CFTR Expression and ER-Associated Degradation in Yeast

Cited by 29 publications

References 3 publications

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

The Thiazide-sensitive NaCl Cotransporter Is Targeted for Chaperone-dependent Endoplasmic Reticulum-associated Degradation

Characterization of anERADGene asVPS30/ATG6Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble Z Variant of Human α-1 Proteinase Inhibitor (A1PiZ) and Another for Aggregates of A1PiZ

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