Figure 5.

Elution profiles of Arp1p mutants when overexpressed. Strains harboring mutant Arp1p were examined by gel filtration and Arp1p was detected by immunoblot with anti-Arp1p antibody. Fraction size is 1 ml. Under these conditions, we are likely examining only the Arp1p filament and capping subunits (see main text). The panels compare elution of wild-type Arp1p with that of Ts^– and lethal alleles, overexpressed from a 2μ plasmid. For wild-type, 10-fold less protein was examined. The Ts^– of arp1-35 and the lethal allele arp1-115 are fully rescued under these conditions, whereas the lethal allele arp1-36 is not rescued by expression to wild-type steady-state levels. Despite these phenotypic differences, all show the same peak elution. Lethal allele arp1-104, which is partially rescued by overexpression, has a notably delayed elution compared with the other mutants. Cytosols were prepared from strains transformed with a 2μ plasmid carrying the same allele as integrated at ARP1. Calibration standards were Void, 6.8 ml, thyroglobulin (669 kDa), 11.0 ml; ferritin (440 kDa), 13.0 ml; aldolase (158 kDa), 14.3 ml; ovalbumin (43 kDa), 15.6 ml; and acetone, 19.7 ml.