Expression and processing of proteins encoded by the Saccharomyces retrotransposon Ty5.
Retroelements (retrotransposons and retroviruses) have two genes in common: gag, which specifies structural proteins that form a virus or virus-like particle, and pol, which specifies catalytic proteins required for replication. For many retroelements, gag and pol are present on separate reading frames. Their expression is highly regulated, and the ratio of Gag to Pol is critical for retroelement replication. The Saccharomyces retrotransposon Ty5 contains a single open reading frame, and we characterized Gag and Pol expression by generating transpositionally active Ty5 elements with epitope tags at the N terminus or C terminus or within the integrase coding region. Immunoblot analysis identified two Gag species (Gag-p27 and Gag-p37), reverse transcriptase (Pol-p59), and integrase (Pol-p80), all of which are largely insoluble in the absence of urea or ionic detergent. These proteins result from proteolytic processing of a polyprotein, because elements with mutations in the presumed active site of Ty5 protease express a single tagged protein (Gag-Pol-p182). Protease mutants are also transpositionally inactive. In a time course experiment, we monitored protein expression, proteolytic processing, and transposition of a Ty5 element with identical epitope tags at its N and C termini. Both transposition and the abundance of Gag-p27 increased over time. In contrast, the levels of Gag-p37 and reverse transcriptase peaked after approximately 14 h of induction and then gradually decreased. This may be due to differences in stability of Gag-p27 relative to Gag-p37 and reverse transcriptase. The ratio of Ty5 Gag to Pol averaged 5:1 throughout the time course experiment, suggesting that differential protein stability regulates the amounts of these proteins.
Irwin
P A
PA
Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011-3260, USA.
Voytas
D F
DF
D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015685:Fusion Proteins, gag-pol / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D018626:Retroelements / Q000235:genetics / Q000502:physiology*; D012440:Saccharomyces / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D015685:Fusion Proteins, gag-pol; D012333:RNA, Messenger; D018626:Retroelements