Figure 2.

O-mannosylation of Δgpαf in vitro is stimulated by ATP and cytosol and requires export-proficient Sec61p. Left, in vitro translated, [³⁵S]methionine-labeled pΔgpαf was translocated into wild-type yeast microsomes, and the washed membranes were incubated at 24°C for the indicated periods of time (minutes) in the presence of ATP and an ATP-regenerating system, cytosol or both. At the end of the incubation period, samples were TCA precipitated and analyzed by SDS-PAGE and autoradiography. Δgpαf and mΔgpαf were quantified using a phophorimager and expressed as percentages of Δgpαf at 0 min. Note that a fraction of Δgpαf is degraded after 60 min, even in the absence of cytosol; the degree of cytosol-independent degradation varies between microsome preparations and is likely due to proteasomes associated with the cytoplasmic faces of the microsomes. Middle, samples contained SEC61 wild-type or the indicated mutant microsomes and were incubated for 20 min in the presence of ATP and cytosol before TCA precipitation. Right, samples contained wild-type microsomes and were incubated with ATP and either wild-type (PRE) or proteasome-mutant (pre1pre2) cytosol for 0 or 20 min before TCA precipitation.