(A–D) Complementation of cdc3-124
ts strain at 36°C by expression of mutant profilins from the weakest
nmt-1 promoter in pRep81 plasmid in the absence of thiamine. (A) Actin
binding mutants. (B) Actin binding mutants, wild-type profilin (WT),
mutant profilin (E42K) for cdc3-124 strain, and
structure stability control mutant C89S. (C) Poly-l-proline
binding mutants. (D) Mutants with profound loss of
poly-l-proline binding Y5D, actin binding K81E, and
nucleotide exchange activity Y79R, mutant with increased nucleotide
exchange activity K81F, ts mutant (E42K), wild-type (WT) profilin,
vector control as well as cdc3-124 strain. (E)
Immunoblots to measure the expression levels of mutant
profilins in cdc3-124 ts strain shifted to 36°C for
4 h. Approximately 25 μg of protein was loaded in each lane.
Table 1 lists the quantitation of these results. No profilin was
detected in the cdc3-124 strain without transformation
or when transformed with mutant E42K.