Fig. 2. Progression and maturation of CPY at non-permissive temperature is promoted by MsNSF. (A) Expression of MsNSF in sec18-1 removes the transport block for CPY. Cells were grown to a density of 1 A₆₀₀ unit ml^-1 in minimal medium lacking methionine and uracil, supplemented with 3 % galactose and 200 µmol l^-1 (NH₄)₂SO₄ for 2 h (A) or 6 h (B,C) to induce expression. Cells were resuspended in fresh medium (lacking sulfate), incubated at 33 °C for 30 min and labeled with trans-³⁵S for 30 min at 33 °C. Cells (10 A₆₀₀ units) were removed at the indicated time intervals and analyzed by CPY immunoprecipitation (IP). Wild-type (B) and MsNSF (C) cells were induced for 6 h before temperature-shift and pulse-chase, and CPY IP was done as in A. Images from three independent experiments were digitized from various exposures and analyzed by NIH image. The CPY maturation index (MI) was calculated from quantified values as [mCPY/(p1+p2+mCPY)]x100. The coefficient of variation was <6 %. Also shown in C is MsNSF (83 kDa) immunoprecipitated with -MsNSF antibody after CPY-IP. The strains used are the same as in Fig. 1 (see Table 1 for genotypes). The relevant portions of autoradiographs are shown. p1 (67 kDa), p2 (69 kDa) and mCPY (61 kDa) refer to ER, Golgi and vacuolar forms of CPY, respectively.