Ubiquitylation of nascent globin chains in a cell-free system.
The ubiquitin/proteasome pathway for degradation of completed and nascent globin chains was evaluated using a cell-free in vitro coupled transcription/translation assay. No decrease in radiolabeled globin chains was observed when ubiquitin, energy regenerating source (or ATP), and E1 and E2 enzymes were added 30 min after the start of translation when globin chain synthesis had plateaued. In contrast, the addition of these components prior to the start of translation resulted in no radiolabeled globin chains after 30 min. The loss of radiolabeled globin chains was dependent on ATP concentration; the higher the concentration, the less the radiolabeled globin chains formed. Prior to the initiation of transcription/translation, cell extract was preincubated with the proteasomal inhibitor MG132 in the absence of globin chain expression vector after which ubiquitin-protein isopeptidase inhibitor, Ubal, and expression vector were added in the presence of 1.5 mm ATP. Thereafter, radiolabeled monoubiquitylated and multiubiquitylated globin chains with few unmodified globin chains were formed. Our results suggest that polyubiquitylated globin chains are localized to the polysomal fractions. These results suggest that nascent globin chains are potential targets for ubiquitylation and deubiquitylation during or soon after translation and that ATP levels play a role in the balance between polypeptide synthesis and degradation.
Adachi
Kazuhiko
K
The Children's Hospital of Philadelphia, Division of Hematology and University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104, USA. adachi@email.chop.edu
Lakka
Vinaysagar
V
Zhao
Yi
Y
Surrey
Saul
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002474:Cell-Free System / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D005914:Globins / Q000378:metabolism*; D006801:Humans; D014176:Protein Biosynthesis; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes; D044763:Ubiquitin-Conjugating Enzymes
D025801:Ubiquitin; D000255:Adenosine Triphosphate; D005914:Globins; D044763:Ubiquitin-Conjugating Enzymes; D010450:Endopeptidases; D044764:Ubiquitin-Activating Enzymes