Fig. 3. Restoration of intra-Golgi and post-Golgi trafficking by MsNSF expression in vivo. (A) Cells were grown in induction medium for 4 h, shifted to 32°C for 30 min and labeled for 30 min. Cell and medium portions were separately precipitated with TCA and analyzed by immunoprecipitation with anti- factor antibody. After 30 min of chase time at 32°C, cells were shifted to 24°C to follow the blocked forms to maturation. Cells were derived from EGY1181-5 (sec18-1) (Table 1), ASHY1181-1 (vector only) (lanes 1-4), and ASHY 1181-2 (P_GAL-MsNSF) (lanes 5-8). During 0 min chase a substantial amount of -factor already resides in the Golgi (lane 5) in MsNSF cells, and this is rapidly matured and secreted (lane 6, 30min chase). In sec18-1 cells the ER form predominates all through the chase time (compare lanes 1 and 2), and is only processed more slowly when returned to the permissive temperature (30 and 60 min chase at 24°C, lanes 3 and 4). The mature peptide was not immunoprecipitated with this antibody. Thus maturation of -factor is inferred from the disappearance of the Golgi (G)-modified form during the chase. ER, G and pG denote the expected positions of ER, Golgi and post-Golgi forms of -factor. The positions of molecular mass markers (kDa) are shown on the left. (B) Analysis of proteins secreted into the medium at restrictive temperatures. Expression of MsNSF in sec18-1 mutants restores transport of proteins secreted into the medium at the restrictive temperature of 37°C. The band at 150 kDa corresponds to an extensively O-glycosylated heat shock protein, HSP150. Wild-type (RSY 248, lane 1) or cells derived from sec18-1 (as mentioned in Fig. 1) were incubated at 37°C for 15 min, labeled and chased for 30 min each at 37°C. Arrows indicate seven predominant proteins that are secretion-blocked in sec18-1 cells. Medium from 0.5 A₆₀₀ cell equivalents was analyzed by autoradiography. Cell pellets were immunoprecipitated with MsNSF antibody to confirm the expression of MsNSF (bottom). (C) Immunoblot of secreted HSP150 at 37°C. Cells were grown overnight in rich medium (supplemented with 3% galactose) to a density of 1 A₆₀₀ unit ml^-1. Cells (25 A₆₀₀ units) were concentrated and washed twice in water and once in medium and resuspended in prewarmed medium (37°C) to a density of 10 A₆₀₀ units ml^-1. Cells (1 ml) were incubated at 37°C for 30 min and equal portions (0 min chase, odd-numbered lanes) were transferred to NaN₃-NaF (20 mmol l^-1) on ice. The remainder of the culture was washed in prewarmed medium to remove pre-existing proteins and resuspended in prewarmed medium containing cycloheximide (20 µg ml^-1); proteins were chased for 30 min (even-numbered lanes). Samples equivalent to 3 A₆₀₀ units of cells were analyzed by immunoblotting with HSP150 antibody. WT, wild type.