We used several diutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae. Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition. The Ty protease is required for processing of both TyA and TyB proteins. Mutations in the protease resulted in the synthesis of morphologically and functidnafly aberrant Ty viruslike particles. The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro. Ty RNA was present in the mutant particles, but at very low levels. Transposition of a genetically tagged element ceased when the protease domain was mutated, demdnstrating that Ty protease is essential for transposition. One of these mutations also defined a segment of TyB encoding an active reverse trnstriptase. These results indicate that the Ty protease, like its retroviral cohnterpart, plays an important role in particle assembly, replication, and transposition of these elements.

Using antibodies directed against the TYBI protein of the transpositionally competent retrotransposon Tyl-H3, we have identified three mature proteins of 23, 60, and 90 kDa and processing intermediates of 140 and 160 kDa that are derived from the 190-kDa TYAI-TYBI polyprotein. Mature proteins and variable amounts of the precursors cofractionate with Ty viruslike particles. The map locations and precursor-product relationships of mature TYBI polypeptides suggest that p23 is Tyl protease, p90 is integrase, and p60 contains reverse transcriptase and RNase H. Immunoprecipitation and immunoblot analyses of Tyl proteins show that p190 is cleaved to form p160. The p160 intermediate is cleaved to form p23 and p140, and p140 is cleaved to form p90 and p60. Processing of TYBI proteins is dependent on Tyl protease. Immunoblot analysis of TYB proteins from different Tyl isolates reveal that correct processing of TYBI proteins is a characteristic of functional Tyl elements, whereas aberrant processing is a common defect found in transposition-incompetent elements.

We used several mutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae. Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition. The Ty protease is required for processing of both TyA and TyB proteins. Mutations in the protease resulted in the synthesis of morphologically and functionally aberrant Ty viruslike particles. The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro. Ty RNA was present in the mutant particles, but at very low levels. Transposition of a genetically tagged element ceased when the protease domain was mutated, demonstrating that Ty protease is essential for transposition. One of these mutations also defined a segment of TyB encoding an active reverse transcriptase. These results indicate that the Ty protease, like its retroviral counterpart, plays an important role in particle assembly, replication, and transposition of these elements.