Induction of the stress protein Grp75 by amino acid deprivation in CHO cells does not involve an increase in Grp75 mRNA levels.
The induction of the stress protein Grp75 in response to amino acid deprivation of Chinese Hamster Ovary cells was characterised using a specific monoclonal antibody. A 2-fold increase in the Grp75 protein content occurred over a period of 5-10 h after incubation of the cells in amino acid-free medium. A partial induction was obtained when either all non-essential amino acids or all essential amino acids were omitted from the medium indicating a broad-specificity response. Deletion of the single amino acids tryptophan, histidine or phenylalanine from otherwise complete medium also produced a partial induction of the protein. The increase in the level of Grp75 was completely blocked by cycloheximide, but only partially blocked by the inhibitors of mRNA synthesis actinomycin D and alpha-amanitin. A specific cDNA probe for Grp75 was generated by PCR and used to quantify mRNA levels. No increase in Grp75 mRNA was observed during the induction of the protein indicating that the primary regulation of Grp75 expression was not at the transcriptional level. These results contrast with the large increase in asparagine synthetase mRNA which has been shown to occur during amino acid deprivation, and indicate that cells respond to this form of stress by more than one mechanism.
Heal
R D
RD
Department of Biochemistry, School of Medical Sciences, Bristol, UK.
McGivan
J D
JD
D016475:3T3 Cells; D000596:Amino Acids / Q000172:deficiency*; D000818:Animals; D001217:Aspartate-Ammonia Ligase / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D015342:DNA Probes; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis*
D000596:Amino Acids; D015342:DNA Probes; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C018551:glucose-regulated proteins; D001217:Aspartate-Ammonia Ligase