Fus1 is on the external surface of fertilization tubules in activated
wild-type mt+ gametes and in a discrete patch on the external surface of
unactivated wild-type mt+ gametes. (A) Fluorescence micrograph of activated
gametes not permeabilized before immunolocalization with the Fus1 antibody. (B
and C) Activated wild-type mt+ gametes incubated with 0% (B) or 0.5% trypsin
(C). The insets for B and C each show control (B) and trypsin-treated (C)
samples dual labeled with anti-Fus1 antibody (green) and Alexa 546-phalloidin
(red). (D) Activated wild-type mt+ gametes incubated with 0.05% trypsin were
analyzed for Fus1 by immunoblotting (top). Identical samples also were
immunoblotted for CALK, an intracellular protein (bottom). In each lane, 2
× 107 cells were loaded. (E and E′) Corresponding
micrographs of unactivated wild-type mt+ gametes dual labeled with the
anti-Fus1 antibody (E, green) and the actin-specific fluorochrome, Alexa
546-phalloidin (E′, red). (F) Unactivated fus1 mt+ gametes
incubated with the anti-Fus1 antibody. (G) Indirect immunofluorescence image
of unactivated nonpermeabilized wild-type mt+ gametes stained with the
anti-Fus1 antibody. (H and I) Anti-Fus1 indirect immunofluorescence images of
control (H) and 0.5% trypsin-treated (I) unactivated wild-type mt+ gametes.
(J) Anti-Fus1 immunoblot of unactivated wild-type mt+ gametes treated with
0.05% trypsin (top). The lower panel shows an anti-CALK immunoblot of
identical samples.