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Abstract:
Spir and formin (FMN)-type actin nucleators initiate actin
polymerization at vesicular membranes necessary for long range vesicular
transport processes. Here we studied in detail the membrane binding
properties and protein/protein interactions that govern the assembly of
the membrane-associated Spir·FMN complex. Using biomimetic membrane
models we show that binding of the C-terminal Spir-2 FYVE-type zinc
finger involves both the presence of negatively charged lipids and
hydrophobic contributions from the turret loop that intrudes the lipid
bilayer. In solution, we uncovered a yet unknown intramolecular
interaction between the Spir-2 FYVE-type domain and the N-terminal
kinase non-catalytic C-lobe domain (KIND) that could not be detected in
the membrane-bound state. Interestingly, we found that the
intramolecular Spir-2 FYVE/KIND and the trans-regulatory
Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore
characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions
in living cells by fluorescence cross-correlation spectroscopy. The data
corroborate a model according to which Spir-2 exists in two different
states, a cytosolic monomeric conformation and a membrane-bound state in
which the KIND domain is released and accessible for subsequent Fmn-2
recruitment. This sequence of interactions mechanistically couples
membrane binding of Spir to the recruitment of FMN, a pivotal step for
initiating actin nucleation at vesicular membranes.
