ER-associated complexes (ERACs) containing aggregated cystic fibrosis transmembrane conductance regulator (CFTR) are degraded by autophagy.
The ubiquitin-proteasome pathway and autophagy are the two major mechanisms responsible for the clearance of cellular proteins. We have used the yeast Saccharomyces cerevisiae as a model system and the cystic fibrosis transmembrane conductance regulator (CFTR) as a model substrate to study the interactive function of these two pathways in the degradation of misfolded proteins. EGFP-tagged human CFTR was introduced into yeast and expressed under a copper-inducible promoter. The localization and degradation of EGFP-CFTR in live cells were monitored by time-lapse imaging following its de novo synthesis. EGFP-CFTR first appears within the perinuclear and sub-cortical ER and is mobile within the plane of the membrane as assessed by fluorescence recovery after photobleaching (FRAP). This pool of EGFP-CFTR is subsequently degraded through a proteasome-dependent pathway that is inhibited in the pre1-1 yeast strain defective in proteasomal degradation. Prolonged expression of EGFP-CFTR leads to the sequestration of EGFP-CFTR molecules into ER structures called ER-associated complexes (ERACs). The sequestration of EGFP-CFTR into ERACs appears to be driven by aggregation since EGFP-CFTR molecules present within ERACs are immobile as measured by FRAP. Individual ERACs are cleared from cells through the autophagic pathway that is blocked in the atg6Delta and atg1Delta yeast strains defective in autophagy. Our results suggest that the proteasomal and the autophagic pathways function together to clear misfolded proteins from the ER.
Fu
Lianwu
L
Department of Cell Biology, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham, AL 35294, USA. lianwufu@uab.edu
Sztul
Elizabeth
E
D001343:Autophagy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D046529:Microscopy, Electron, Transmission; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection
C485184:enhanced green fluorescent protein; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex