Figure 3.

(A) The overall secretory pathway is not impaired in tlg2Δ cells. FHER001-3B (WT) and FHER001-5A (tlg2Δ) transformed by p195gF (2μ URA3 GAL-FUR4) were grown in 2% lactate minimal medium to an A₆₀₀ of 0.4. Galactose (4%) was added to induce uracil permease expression. Uracil uptake activity was measured at various time intervals. The lower uracil permease specific activity in mutant relative to wild-type cells accompanied by a parallel lower amount of immunodetected permease probably resulted from the slower growth of these cells in galactose medium. Transformation of disrupted cells with centromeric plasmid carrying TLG2 complemented the slower growth and the reduction in permease (our unpublished results). (B and C) Maturation of vacuolar proteins CPY and and ALP in wild-type and tlg2Δ cells. (B) FHER001-3B (WT) and FHER001–5A (tlg2Δ) cells were pulse labeled with [³⁵S]methionine and chased with nonradioactive methionine for the indicated times. CPY was immunoprecipitated from samples taken at various times. The immunoprecipitates were analyzed on 7.5% polyacrylamide/0.1% SDS gels and visualized by fluorography. p1, ER form; p2, Golgi form; m, mature vacuolar form. (C) FHER001-3B (WT), FHER001-5A (tlg2Δ), and SL (sec18) cells were collected in the exponential phase in YPD medium at 30°C (24°C for SL) and either left at permissive temperature or transferred to 37°C for 2 h. Protein extracts were prepared. Aliquots corresponding to 4 × 10⁶ cells were analyzed for CPY and ALP by Western immunoblotting. In sec18 cells, mature proteins synthesized before the temperature block, and ER-accumulated forms of CPY (p1) and ALP (pro) synthesized after exposure at the restrictive temperature, were visible.