Investigating Functional Motifs within the N- Terminal Domain of Novel Actin Binding Proteins NET1A and NET2A

Abstract

The NET proteins are a novel family of actin binding proteins that all share a homologous n-terminal actin binding domain (ABD). The aims of this project were to probe potential functional domains within this ABD by creating mutants of NET1A and NET2A as representative family members. Mutants were designed with the aim of disrupting f-actin binding. To identify possible sites for mutation, the ABDs of the NET family were analysed to look for residues that were homologous to all members except NET3B, which has been shown not to bind actin. Once potential residues were identified, point mutants were designed and cloned alongside an ABD deleted construct. The clones were then expressed in a dominant negative approach using transient expression techniques.

Infiltrations of NET1A mutants into Nicotiana benthamiana leaves showed changes in the localisation and behaviour of the protein relative to wild type. These included a potential association between NET1A and the ER, identification of a potential single residue in the ABD that contacts f-actin and whose mutation reduces NET1A’s f-actin affinity and the disruption to the association between both the ER and f-actin, and the plasma membrane. Results from these infiltrations have led to the development of a model that suggests potential functions for discrete domains within NET1A. This model will be useful for directing future experiments that aim to identify proteins that NET family interact with and perhaps identify common functions for these novel actin binding proteins.