Proteolytic processing of pol-TYB proteins from the yeast retrotransposon Ty1

Abstract

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.

Retroviral-like determinants and functions required for dimerization of Ty1 retrotransposon RNA

“…During or soon after VLP assembly, the precursor Ty1 Gag polyprotein (Gag-p49) undergoes a single C-terminal cleavage that results in mature Gag-p45 ( Fig. 1b) [31][32][33][34]. Despite the lack of sequence homology, Ty1 Gag is functionally related to retroviral Gag proteins as it comprises the capsid of VLPs and mediates critical RNA transactions during the retrotransposon replication cycle [21,28].…”

The Ty1 LTR-Retrotransposon of Budding Yeast, Saccharomyces cerevisiae

“…The Gag-Pol precursor must be cleaved at the Gag/PR site for subsequent cleavages of the Gag-Pol precursor or p49-Gag precursor to occur [125]. Pulse-chase experiments of pGTy1 expression and mutagenesis of PR cleavage sites support semi-ordered processing of the p49-Gag and p199-Gag-Pol precursor proteins [124,125]. Mutations in Ty1 that abolish PR activity result in VLPs with significant reverse transcriptase activity when an exogenous primer and template are supplied, but reverse transcription of the endogenous template is undetectable, perhaps because VLPs produced from a PR-defective element contain significantly less Ty1 gRNA, and Ty1 RNA dimerization is reduced [116,126].…”

“…Ty1 protease (PR) is a 20 kDa monomeric aspartyl protease encoded by the C-terminus of the GAG ORF and N-terminus of the POL ORF. Processed p20-PR is very difficult to detect, even when pGTy1 is expressed [124]. Ty1 PR activity is required for all cleavages of Ty1 Gag and Gag-Pol proteins and for retrotransposition activity [124][125][126].…”

“…Processed p20-PR is very difficult to detect, even when pGTy1 is expressed [124]. Ty1 PR activity is required for all cleavages of Ty1 Gag and Gag-Pol proteins and for retrotransposition activity [124][125][126]. The p199-Gag-Pol precursor has three distinct cleavage sites at the Gag/PR (RAH/NVS), PR/IN (TIN/ NVH) and IN/RT (LIA/AVK) junctions, which were defined by sequencing of various termini of mature Ty1 proteins and mutagenesis [125,127,128].…”

Virus-like particle analysis in yeast homogenate using a laser light-scattering assay

“…The biological system studied by DLS in this article is a noninfectious multimeric protein of a particulate nature, termed virus-like particle (VLP), which is approximately 80 nm in diameter and is produced intracellularly by the yeast transposon Ty1 in Saccharomyces cerevisiae. The naturally occurring particles contain a combination of structural monomers (Gag protein, ≈50 kDa) and catalytically important fusion proteins (Gag-pol fusion protein, ≈190 kDa; Garfinkel et al, 1991;Müller et al, 1987). The Ty1 gene sequences (gag and pol) can be engineered to produce particles of Ty1 Gag-foreign protein fusions.…”