Immunofluorescence localization of Sec4p in
different sec2 alleles and a
sec2-78,6-4 double mutant after a short shift to the restrictive temperature. Cells were
grown overnight in YP dextrose medium at 25°C and
then shifted to 37°C for 10
min, fixed, and labeled with
αSec4 antibody. sec12-4,
NY738; sec2-41, NY132;
sec2-59, NY27; sec2-78,
NY1529; wild-type, NY10;
sec6-4, NY779; sec2-78,6-4,
NY1530. In sec2-41, sec2-59,
and sec2-78, Sec4p, immunostaining was concentrated in
the bud at 25°C but was diffuse already after 10-min
shift to the restrictive temperature. In the ER-to-Golgi
mutant sec12-4, diffuse immunolabeling was also already observed after 10 min
at 37°C. Both in wild-type
and sec6-4, Sec4p immunolabeling was concentrated at
the bud both at 25°C and after 10 min at 37°C. The double mutant was constructed
as described in Materials and
Methods. sec2-78, sec2-78,6-4
shows labeling in the buds at
the permissive temperature
that is no longer detected after 10 min at the restrictive
temperature.