Deletion of END4 constitutively
blocks endocytosis. (A) The complete END4 ORF on an
AatII fragment was replaced with the prototrophic marker
HIS3. The resulting EcoRI fragment was
used to delete END4 by integration/replacement. B,
BamHI; R, EcoRI; A, AatII;
X, XhoI; H, HindIII. (B)
Receptor-mediated internalization of α-factor is blocked at both
24°C (solid symbols) and 37°C (open symbols) in RH3391 cells cured
of pGP6 (end4Δ, squares), whereas RH3393 cells
(END4, circles) show no defect at either temperature.
(C) End4p-specific antiserum generated against a C-terminal peptide
specifically recognizes a single band with an apparent mobility of
∼116 kDa that is absent in strain RH3391 cured of pGP6
(end4Δ) extracts but more prominent in extracts
prepared from strains expressing multiple copies of the gene
(2μEND4). (D) In contrast to strain RH2887
(END4), strain RH3391 cured of pGP6
(end4Δ) does not accumulate LY in the vacuole after
incubation with the dye at 24°C for 1 h. Bar, 5 μm.