FIG. 5.

Degradation of CY028 cutinase. S. cerevisiae cells expressing CY028 were incubated at 30°C for 30 min on YNB medium containing 2% galactose with or without the proteasomal inhibitor clasto-lactacystin-β-lactone at a concentration of 25 μM. The cells were pulse-labeled for 1 min with 150 μCi of [³⁵S]Met/Cys. After washing, the cells were chased by taking samples at different times. The samples were then subjected to immunoprecipitation with anti-cutinase and loaded on SDS-PAGE gels, and autoradiography was performed. (A) Scanning values obtained with a personal densitometer from Molecular Dynamics. At 5 min no degradation was found after treatment with the proteasomal inhibitor (•), in contrast to the untreated S. cerevisiae CY028-expressing cells (⧫). After deletion of the UBC7 gene, degradation was delayed (▪. All values were normalized by using the cutinase level at zero time. (B) Corresponding autoradiographs after immunoprecipitation on the SDS-PAGE gels for different times (0, 10, 30, 60, and 300 s).