FIG. 5.

Overproduction of Tim23p causes a defect in protein import. The tim23-1 strain KRR131 carrying the GAL1-tim23C plasmid pKR17 was grown to an OD₆₀₀ of 0.5 at the permissive temperature, 23°C, in selective medium containing 2% raffinose and then split into two aliquots. Galactose (GAL) was added to one aliquot to a final concentration of 4%, and the other sample remained in raffinose (RAF) medium. After 4 h, an aliquot of each culture was removed, the cells were pulse-labeled for 5 min with [³⁵S]methionine (13), and further labeling was stopped by the addition of 1 mM sodium azide. Total proteins were extracted from cells and immunoprecipitated with antiserum to F₁β, the β subunit of F₁-ATPase (13, 78). Precipitates were analyzed by SDS-PAGE followed by fluorography. The precursor (p) and mature (m) forms of F₁β are indicated.