Stress-induced retrotranslocation of clusterin/ApoJ into the cytosol.
Clusterin is a usually secreted glycoprotein with chaperone properties. Recently, it has been suggested that clusterin isoforms reside in the nuclear and cytosolic compartments of human cell types, where they can influence various cellular programs including DNA repair, transcription and apoptosis. Several mechanisms have been proposed to explain this atypical location, including alternative transcription initiation and alternative splicing. However, none of these have been unequivocally established as occurring in live cells. Here we provide direct experimental evidence that in live intact cells, under certain stress conditions, clusterin can evade the secretion pathway and reach the cytosol. This was demonstrated using several complementary approaches. Flow cytometry and selective permeabilization of U251 cell membranes with digitonin allowed detection of cytosolic clusterin in stressed U251 cells. In addition, a stringent enzymatic assay reliant upon the exclusively cytosolic deubiquitinase enzymes confirmed that clusterin synthesized with its hydrophobic secretion signal sequence can reach the cytosol of U251 cells. The retrotranslocation of clusterin is likely to occur through a mechanism similar to the endoplasmic reticulum (ER)-associated protein degradation pathway and involves passage through the Golgi apparatus. We also report that the ER-associated ubiquitin ligase Hrd1/synoviolin can interact with, and ubiquitinate clusterin. The possible biological functions of these novel behaviours of clusterin are discussed.
Nizard
Philippe
P
UMR6026 CNRS Université de Rennes1, Intracellular Protein Homeostasis, IFR 140, Campus de Beaulieu, Bat. 13, 35042 Rennes cedex, France.
Tetley
Susanne
S
Le Dréan
Yves
Y
Watrin
Tanguy
T
Le Goff
Pascale
P
Wilson
Mark R
MR
Michel
Denis
D
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D019556:COS Cells; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D051152:Clusterin / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000235:genetics; D011189:Potassium Chloride / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
C495242:CLU protein, human; D002614:Chelating Agents; D051152:Clusterin; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009842:Oligopeptides; D061988:Proteasome Inhibitors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C073762:lysyl-aspartyl-glutamyl-leucine; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D004533:Egtazic Acid; D011189:Potassium Chloride; D010450:Endopeptidases; D046988:Proteasome Endopeptidase Complex; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde