Aip1p Accelerates Actin Filament Disassembly
in a Cofilin Dependant Manner
Shown below is an actin filament pelleting
assay: actin purified from yeast was induced to polymerize with
salt and magnesium in the presence (lanes 3-7) or absence (lanes
1 and 2) of Aip1p and in the presence of different amounts of
cofilin. The filaments are then pelleted and the supernatant versus
the pellet is sampled in a coomassie stained gel to quantify the
amount of actin present in the filamentous form. As can bee seen
in lane 1, actin alone efficiently forms filaments. The addition
of cofilin at stoichiometric amounts (lane 2) induces a slight
decrease in the amount of F-actin due to the cofilin induced increase
in off rate from the minus ends of the filaments and a weak filament
severing activity. Note the cofilin is largely present in the
pellet fraction as it binds actin filaments 1:1 with the actin
subunits. The addition of Aip1p at a 1:1 stoichiometry causes
a large decrease in actin from the pellet phase and this effect
is dependent on cofilin, little effect of Aip1p is observed once
the cofilin is dropped down to 1/5 the amount of actin. This assay
shows that Aip1p can act to destabilize actin filaments but only
actin filaments that are optimally decorated with cofilin.
