Rapid turnover of adenovirus E1A is determined through a co-translational mechanism that requires an aminoterminal domain.
The product of the adenovirus E1A 13S mRNA can both stimulate and repress the expression of certain viral and cellular genes. As with several other regulatory proteins, E1A has a short half-life, approximately 40 min. Although this short half-life is observed in cells expressing the E1A gene, it is not the case with cells injected with E1A protein, where its half-life is very long, generally greater than 15 h. We have sought to reconcile these apparent differences in E1A stability. Using Xenopus oocytes, we find that E1A exhibits its characteristic short half-life when it is synthesized from injected mRNA while it has a very long half-life when it is injected as a protein synthesized originally in Escherichia coli or reticulocyte lysates. In order to delineate the amino acids responsible for rapid E1A turnover, several deletion mRNAs were constructed, injected into oocytes, and E1A half-life determined. Carboxyl-terminal deletions and an internal deletion of residues 38-86 failed to increase the half-life of E1A. In contrast, amino-terminal deletions of 70 and 14 residues resulted in very stable E1A proteins (t1/2 greater than 20 h). Furthermore, deletion of the second amino acid, an arginine, resulted in a stable E1A protein. The amino-terminal region of E1A was able to induce the rapid turnover of a normally stable protein, beta-globin, in oocytes injected with an E1A-globin chimeric mRNA. This E1A-induced instability of globin was abolished, however, when the protein was first synthesized in reticulocyte lysates and then injected into oocytes. The amino-terminal region of E1A is also important in governing halflife in adenovirus-infected HeLa cells. These results demonstrate that the half-life of E1A is established cotranslationally through a mechanism involving sequences within the amino-terminal 37 residues.
Slavicek
J M
JM
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Jones
N C
NC
Richter
J D
JD
D017456:Adenovirus Early Proteins; D000595:Amino Acid Sequence; D000818:Animals; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D014176:Protein Biosynthesis; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014982:Xenopus laevis
D017456:Adenovirus Early Proteins; D004268:DNA-Binding Proteins; D009856:Oncogene Proteins, Viral; D014157:Transcription Factors