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Fig. 1. Isolation of Drosophila Rabenosyn-5 homolog.
(A,B) GFP-Vas localization in wild-type (A) and C241
GLC (B) oocytes. Enlarged images of the bracketed regions are shown in the
right panels. Mutant clones were identified by the lack of nuclear GFP signals
in the germline. (C) Domain structure of CG8506 and its orthologs. The
positions of the C2H2 and FYVE (Fab1p, YOTB, Vac1p and EEA1) zinc fingers,
coiled-coil domain and NPF (Asn-Pro-Phe) motif are indicated. The
C241 mutant contains a premature stop codon at the residue 315
(asterisk). (D) Western blot of ovarian extracts from wild-type and
C241 heterozygotes with rabbit and rat polyclonal anti-Rbsn-5
antisera. A single band of the expected size for Rbsn-5 was detected. A
truncated protein resulting from a premature stop codon (expected size of 35
kD; asterisk) was undetectable in C241/+ lysates. (E)
Drosophila Rbsn-5 specifically interacts with the GTP-bound form of
Rab5. In-vitro-translated Rbsn-5 was incubated with GST-Rab5, GST-Rab11 and
GST-Rab7 in the presence of GDP or GTP
S. Bound proteins were analyzed
by western blot using anti-Rbsn-5 antisera. (F,G)
Auto-fluorescent granules derived from endocytosed yolk were detected in
wild-type oocytes (F) but not in rbsn-5- oocytes (G).
(H,I) Incorporation of the FM4-64 dye by wild-type (H) and
rbsn-5- (I) oocytes. No internalization of FM4-64 dye was
detected in rbsn-5- oocytes. Anterior is to the left,
posterior is to the right in all images. Scale bars: 20 µm.
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