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.
