N terminus of End4p is essential for actin
organization and endocytosis. (A) Mutant end4 alleles
devoid of the indicated regions (open space in the schematic
representation) were generated as described in MATERIALS AND METHODS.
Pro, proline-rich sequence; Q, glutamine-rich sequence; L, putative
leucine-zipper; coil1 and coil2, putative coiled-coil regions 1 and 2,
respectively. Proline at aa 498 divides the region from aa 376 to aa
573 into two separate coiled-coil domains (MATERIALS AND METHODS). The
nomenclature of end4 alleles and the deleted amino acids
are indicated. (B) END4 (section 1, RH3393),
end4Δ (section 2, RH3212),
end4ΔN1 (section 3, RH3769), and
end4Δcoil1 (section 4, RH3395) strains
were streaked onto YPUADT plates and incubated for 2 d at 37°C.
Deletion of the N-terminal part of End4p but not deletion of the
coiled-coil domain impairs growth at higher temperatures. (C)
Receptor-mediated endocytosis of radiolabeled pheromone in
END4 (RH3393) and
end4ΔN1 (RH3769) strains after a 5-min
preincubation at either 24°C (solid symbols) or 37°C (open
symbols). (D) END4 (RH3393) and
end4ΔN1 (RH3769) strains were grown at
24°C and processed for visualization of filamentous actin with
rhodamine-conjugated phalloidin as described in MATERIALS AND
METHODS. Bar, 5 μm.