Processing of the proteins encoded by the Saccharomyces retrotransposon Ty5
Date
2000
Authors
Irwin, Phillip Allen
Major Professor
Advisor
Voytas, Daniel F.
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Retrotransposons and retroviruses have two genes in common - GAG, which encodes structural proteins that form a virus or virus-like particle, and POL, which encodes the enzymatic activities required for replication. For most retroelements, GAG and POL are encoded on separate reading frames. The relative levels of these proteins are carefully regulated, and the GAG to POL ratio is critical for retroelement replication. In contrast, the Saccharomyces cerevisiae retrotransposon, Ty5, encodes a single open reading frame. To characterize GAG and POL expression, we generated several functional Ty5 elements with epitope tags at the N-terminus, C-terminus and within the integrase-coding region. Using antibodies against these epitope tags, we identified several Ty5 proteins that are similar in size to counterparts encoded by other retroelements. These include two GAG proteins (p27GAG and p37GAG), reverse transcriptase (p57RT), and integrase (p661N), all of which are insoluble in the absence of urea or ionic detergents.
A mutation generated in the predicted active site of the Ty5-encoded protease is transpositionally inactive, and a single polyprotein is observed (p18OPoly). This indicates that the Ty5 protease is responsible for polyprotein processing. We followed proteolytic processing during a time course after induction of Ty5 transcription. As the culture approached stationary phase, the abundance of p37GAG reduced significantly until no longer detected. This suggests that cell culture conditions trigger either preferential degradation of p37GAG or processing to the p27GAG form. The change in abundance of p37GAG roughly corresponds to an approximately 2-fold increase in transposition. These changes in protein processing and the increase in transposition are similar to what has been observed previously for the Tf1 retrotransposon of Schizosaccharomyces pombe, which also encodes GAG and POL on a single open reading frame. For Tf1, the changes in protein abundance were considered a mechanism by which GAG/POL stoichiometry is regulated. It remains to be determined if this is the case for Ty5.
Series Number
Journal Issue
Is Version Of
Versions
Series
Academic or Administrative Unit
Type
thesis