Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae.
Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins. To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p. Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells. Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker. Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin beta-lactone. Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment. Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p. Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation. These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae.
Kiser
G L
GL
S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, USA.
Gentzsch
M
M
Kloser
A K
AK
Balzi
E
E
Wolf
D H
DH
Goffeau
A
A
Riordan
J R
JR
D003546:Cysteine Endopeptidases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection
C505032:CFTR protein, human; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex