Processing of TY1 proteins and formation of Ty1 virus-like particles in Saccharomyces cerevisiae

Abstract

We have analysed functional properties of putative proteins encoded by the yeast transposable element, Ty1, by overexpression of TY genes. High-level expression was achieved by appropriate fusion of a Ty sequence, TY9C, to the yeast ADH1 promoter and transformation of yeast cells with this construction. As shown recently by others (Garfinkel et al. 1985; Mellor et al. 1985c) TY overexpression leads to an increase in particle-bound reverse transcriptase activity and to an intracellular accumulation of virus-like particles (Ty-VLPs). We have used a number of deletions in the second open reading frame (TYB) to identify functional domains required for processing and assembly of Ty proteins. Deletions in the TYB region with homology to acid proteases result in overproduction of an unprocessed form of the TYA protein (pro-TYA) which represents the major protein of Ty-VLPs. One particular mutant construction, TY9C-delta 36, led to the accumulation of a particle-bound, 160 kDa protein which cross-reacted with a mouse antiserum raised against purified pro-TYA protein. This supports the hypothesis that TYB is expressed as a TYA/TYB fusion protein which is processed by a TYB-encoded protease activity. Ty-VLPs are formed in the absence of protein processing and even when the TYB gene is not expressed. Thus, we assume that the assembly of Ty particles occurs prior to processing of Ty proteins.

BUD22 Affects Ty1 Retrotransposition and Ribosome Biogenesis in Saccharomyces cerevisiae

“…Most Gag-p45 is derived from proteolytic cleavage of Gag-p49, which is the primary translation product of the GAG gene. A 11 frameshift signal near the 39 end of GAG results in the production of a 199-kDa Gag-Pol fusion protein (Clare et al 1988;Belcourt and Farabaugh 1990), which contains the Gag structural protein, and the enzymes protease (PR), integrase (IN), and reverse transcriptase (RT) (Adams et al 1987;Muller et al 1987;Eichinger and Boeke 1988;Youngren et al 1988;Garfinkel et al 1991). The efficiency of frameshifting helps determine the optimal ratio of Gag and Gag-Pol proteins (estimated at $30:1) that is required for PR function and retrotransposition (Xu and Boeke 1990;Kawakami et al 1993).…”

Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition.

“…The cofractionation of Ty-VLPs, reverse transcriptase activity, a TyB-encoded protein, and genomelength Ty RNA, as well as the ability of this complex to synthesize Ty DNA, suggests that reverse transcription takes place in the particle and that Ty-VLPs are transposition intermediates (20,37). Recently, the major structural proteins of Ty-VLPs have been shown to be derived from the primary product of TyA by proteolytic cleavage (1,39). At least some of these cleavages appear to be mediated by the product of the protease domain of TyB.…”

The Gag polypeptides of the Drosophila 1731 retrotransposon are associated to virus‐like particles and to nuclei

“…1), we had obtained the first evidence for the existence of 1731 Gag-like proteins; indeed, 1731 Gag-fl-galactosidase fusion proteins were found into VLPs-containing, RTactive fractions extracted from Drosophila virilis transfected cells [12]. As Gag proteins from both retrotransposons and retroviruses have been repeatedly proven to be difficult to characterize when only the Mr was considered [24,25], we prepared antibodies directed against the central part of the 1731 Gag protein, assuming that they would allow recognition of gag products in cells and/or in organisms.…”

“…The p32 species is thought to be the primary, unmodified form of the Gag protein and the p40-42 should correspond to a post-translationally modified p32. Such apparent larger sizes of Gag proteins are frequently encountered and were reported for the two yeast retrotransposons Tyl and Ty3 [25,[28][29][30]. However, the nature of the post-translational modification of 1731 Gag is unknown.…”

“…As orderly species of Gag proteins have been described in both retroviruses and retrotransposons [24,25], the appearance of a single immunoreactive 1731 Gag protein in the gradient fractions was surprising. In order to search other forms of 1731 Gag, we concentrated cytoplasmic particles by pelleting a postmitochondrial supernatant through a sucrose cushion, at 150,000 x g for one hour.…”