Preferential perinuclear localization of poly(ADP-ribose) glycohydrolase.
The transient nature of poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, is achieved by the enzyme poly(ADP-ribose) glycohydrolase (PARG) which hydrolyzes the poly(ADP-ribose) polymer into free ADP-ribose residues. To investigate the molecular size and localization of PARG, we developed a specific polyclonal antibody directed against the bovine PARG carboxy-terminal region. We found that PARG purified from bovine thymus was recognized as a 59-kDa protein, while Western blot analysis of total cell extracts revealed the presence of a unique 110-kDa protein. This 110-kDa PARG was mostly found in postnuclear extracts, whereas it was barely detectable in the nuclear fractions of COS7 cells. Further analysis by immunofluorescence revealed a cytoplasmic perinuclear distribution of PARG in COS7 cells overexpressing the bovine PARG cDNA. These results provide direct evidence that PARG is primarily a cytoplasmic enzyme and suggest that a very low amount of intranuclear PARG is required for poly(ADP-ribose) turnover.
Winstall
E
E
Faculty of Medicine, Laval University Medical Research Center, Québec, G1V 4G2, Canada.
Affar
E B
EB
Shah
R
R
Bourassa
S
S
Scovassi
I A
IA
Poirier
G G
GG
D000818:Animals; D002417:Cattle; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
D018076:DNA, Complementary; D011064:Poly Adenosine Diphosphate Ribose; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase