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Ty1 Defect in Proteolysis at High Temperature

Joseph F. Lawler, Jr.,¹ Daniel P. Haeusser,^2, Angie Dull,^2, Jef D. Boeke,¹ and Jill B. Keeney^2*

Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,¹ Department of Biology, Juniata College, Huntingdon, Pennsylvania 16652²

Received 11 July 2001/ Accepted 15 January 2002

Retrotransposition of the Ty1 element of Saccharomyces cerevisiae is temperature sensitive. Transposition activity of Ty1 is abolished at temperatures above 34°C. In this report, we show that the major block to transposition at high temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and the concomitant reduction of reverse transcriptase (RT) activity. Expression of a Ty1 protease construct in Escherichia coli shows that protease enzymatic activity is inherently temperature sensitive. In yeast, Gag processing is only partially inhibited at high temperature, while cleavage of the Gag-Pol polyprotein is completely inhibited. Sites of proteolytic processing are differentially susceptible to cleavage during growth at high temperature. Overall levels of the Gag-Pol polyprotein are reduced at high temperature, although the efficiency of the requisite +1 frameshifting event appears to be increased. RT activity is inherently relatively temperature resistant, yet no cDNA is made at high temperature and the amount of RT activity is greatly reduced in virus-like particles formed at high temperature. Taken together, these results suggest that alterations in Ty1 proteins that occur at high temperature affect both protease activity and RT activity, such that Ty1 transposition is abolished.

Present address: Molecular Cell Biology Program, Washington University School of Medicine, St. Louis, MO 63110.

Present address: Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University, University Park, PA 16801.

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