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. 1998 Feb;18(2):779–789. doi: 10.1128/mcb.18.2.779

FIG. 3.

FIG. 3

Ubiquitination and metabolic stability of Ste6p in a ubc4,5Δ mutant, defective for a pair of ubiquitin-conjugating enzymes. (A) To determine the extent of ubiquitination of Ste6p in wild-type and mutant strains, unlabeled extracts were prepared from a strain containing two plasmids, pSM683 (STE6-HA) and YEp105 (UBI::myc), or the corresponding untagged versions pSM192 (CEN URA3 STE6) and YEp96 (2μm TRP1 UBI::myc). Ste6p-HA was immunoprecipitated from the unlabeled extracts with anti-HA antibodies; immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose. Two separate gels and filters were prepared. One filter was probed with anti-Myc antibodies to detect ubiquitin-conjugated forms of Ste6p-HA, and the other was probed with anti-HA antibodies to assess the total amount of Ste6p-HA in each extract. Extracts in panel A were prepared from SM3126 (pSTE6::HA, vector) in lane 1, SM3127 (pSTE6::HA, pUBI::myc) in lane 2, SM3128 (pUBI::myc, vector) in lane 3, and SM3129 (pSTE6::HA, pUBI::myc, in ubc4Δ ubc5Δ) in lane 4. The position at which Ste6p migrates (ca. 145 kDa) is indicated by a bar, and the 200-kDa size marker is indicated in panel A. (B) To compare the half-life of Ste6p-HA in wild-type and ubc4,5Δ mutant strains, cells were pulse-labeled for 10 min with Express 35S. Strains used were SM3624 (wild type) and SM3628 (ubc4,5Δ). The label was chased for the indicated times (minutes). Cell extracts were immunoprecipitated with anti-HA antibodies to determine the half-life of Ste6p (top) and with anti-Cpy antibodies to examine the time course of Cpy processing (bottom). The p1 (ER), p2 (Golgi), and mature (m; vacuolar) forms are indicated. The kinetics of Ste6p degradation was determined by quantitation of the Ste6p band at each time point (C). Quantitation of Ste6p degradation was performed by PhosphorImager analysis as described in Materials and Methods. Open circles and squares designate the time points for the wild-type (WT) and ubc4,5Δ mutants, respectively. The calculated half-life (t1/2) of Ste6p-HA is indicated.